CN111560354B

CN111560354B - Recombinant novel coronavirus, preparation method and application thereof

Info

Publication number: CN111560354B
Application number: CN202010439738.3A
Authority: CN
Inventors: 杨鹏辉; 王希良; 程晋霞; 王欢; 孙芳
Original assignee: Beijing Maidi Libang Biotechnology Co ltd; Fifth Medical Center of PLA General Hospital
Current assignee: Beijing Maidi Libang Biotechnology Co ltd; Fifth Medical Center of PLA General Hospital
Priority date: 2020-05-22
Filing date: 2020-05-22
Publication date: 2022-07-19
Anticipated expiration: 2040-05-22
Also published as: CN111560354A

Abstract

The invention discloses a recombinant novel coronavirus, a preparation method and application thereof. The present invention first discloses a recombinant virus in which any one of an NS gene of an influenza virus, an HA gene of an influenza virus, and an NA gene of an influenza virus is substituted. The invention further discloses the application of the recombinant virus in preparing products for preventing and/or treating diseases caused by influenza virus and/or SARS-CoV-2. The invention operates SARS-CoV-2 epitope and influenza virus genome from gene level, prepares recombinant SARS-CoV-2 vaccine strain taking influenza virus as carrier based on RG technology, solves the shortage problem of SARS-CoV-2 vaccine, is a new milestone in the field of coronavirus vaccine, and SARS-CoV-2 chimeric vaccine can protect more crowds from influenza virus and SARS-CoV-2.

Description

Recombinant novel coronavirus and preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a novel recombinant coronavirus, and a preparation method and application thereof.

Background

Coronaviruses are a class of enveloped, linear, single-stranded, positive-stranded RNA viruses whose genome is circular or elliptical in shape, with particles of about 60-140nm in diameter. The virion is surrounded by a fat membrane, the surface of which has three glycoproteins: spike glycoprotein (SpikeProtein, S): a globular-rod-shaped protrusion constituting the surface of the virion belongs to the type I transmembrane protein. Can be decomposed into two functional units of S1 and S2 by the S enzyme. S1 is used to promote the binding of virus to host cell receptor, and the binding region (RBD region) of host receptor is combined with receptor, and this region can be combined with ACE2 protein, if the combination is completed, the novel coronavirus will infect human respiratory epithelial cell, and further cause pneumonia; membrane glycoprotein (M): embedding into the viral envelope through three transmembrane domains; envelope protein (enveloppeprotein, E): a transmembrane protein; the S protein determines the host range and specificity of the virus, is an important action site of a host neutralizing antibody, and is a key target point of vaccine design.

The novel coronavirus named SARS-CoV-2 by the International Commission on Virus Classification (ICTV) is a newly discovered virus strain spread in human beings, the symptoms of the novel coronavirus pneumonia infectors can range from common cold to severe pulmonary infection, the initial clinical symptoms of the novel coronavirus pneumonia infectors are not obvious, the infectors cannot pay high attention to the symptoms, the morbidity is high, severe pneumonia is caused in the later period, respiratory distress syndrome (ARDS) caused by inflammatory factor storm causes death, the novel coronavirus pneumonia epidemic situation (COVID-19) caused by the infection of the novel coronavirus (SARS-CoV-2) is extremely similar to the clinical symptoms of SARS-CoV, the novel coronavirus pneumonia epidemic situation (COVID-19) becomes an emergent public health incident (PHEIC) of international concern, great harm is caused to the health, social stability and the national economy of people, and the research and development of emergency vaccines are urgently needed.

Currently, live attenuated influenza vaccines based on Reverse Genetics (RG) of influenza virus are approved to be on the market to show safety and effectiveness, and the influenza virus is spotlighted as a vector for expressing foreign genes. So far, the use of influenza virus as a vector for expressing SARS-CoV-2 virus key protein in the development of recombinant SARS-CoV-2 virus strain has not been reported yet.

Disclosure of Invention

The technical problem to be solved by the invention is how to effectively avoid the shortage problem of SARS-CoV-2 vaccine.

In order to solve the above technical problems, the present invention provides a recombinant virus.

The recombinant virus provided by the invention is prepared according to a method comprising the following steps: co-transfecting host cells with a recombinant plasmid containing a PB2 gene of an influenza virus, a recombinant plasmid containing a PB1 gene of the influenza virus, a recombinant plasmid containing a PA gene of the influenza virus, a recombinant plasmid containing an NP gene of the influenza virus, a recombinant plasmid containing an M gene of the influenza virus, a recombinant plasmid containing an NS gene of the influenza virus, a recombinant plasmid containing an HA gene of the influenza virus and a recombinant plasmid containing an NA gene of the influenza virus, and culturing to obtain the recombinant virus;

any one of the NS gene of the influenza virus, the HA gene of the influenza virus and the NA gene of the influenza virus is replaced: the NS gene of the influenza virus is replaced by a recombinant DNA molecule named as S-RBD + E + M-NS gene or 2S-RBD-NS gene, the HA gene of the influenza virus is replaced by a recombinant DNA molecule named as S-RBD-HA gene or S1-HA gene or 2S-RBD-HA gene, and the NA gene of the influenza virus is replaced by a recombinant DNA molecule named as S-RBD-NA gene or S1-NA gene or 2S-RBD-NA gene;

the S-RBD + E + M-NS gene is a recombinant DNA molecule obtained by inserting the S-RBD + E + M gene between the 375 th nucleotide and the 376 th nucleotide from the 5' end of the open reading frame of the NS gene of the influenza virus and keeping other nucleotides of the NS gene of the influenza virus unchanged;

the S-RBD-HA gene is a recombinant DNA molecule obtained by inserting the S-RBD gene between the 51 st nucleotide and the 52 nd nucleotide from the 5' end of the open reading frame of the HA gene of the influenza virus and keeping other nucleotides of the HA gene of the influenza virus unchanged;

the S-RBD-NA gene is a recombinant DNA molecule obtained by replacing 193 rd to 213 th nucleotides from the 5' end of an open reading frame of the NA gene of the influenza virus with the S-RBD gene and keeping other nucleotides of the NA gene of the influenza virus unchanged;

the 2S-RBD-NS gene is a recombinant DNA molecule obtained by inserting the 2S-RBD gene between the 375 nd nucleotide and the 376 nd nucleotide from the 5' end of the open reading frame of the NS gene of the influenza virus and keeping other nucleotides of the NS gene of the influenza virus unchanged;

the 2S-RBD-HA gene is a recombinant DNA molecule obtained by inserting the 2S-RBD gene between the 51 st nucleotide and the 52 nd nucleotide from the 5' end of the open reading frame of the HA gene of the influenza virus and keeping other nucleotides of the HA gene of the influenza virus unchanged;

the 2S-RBD-NA gene is a recombinant DNA molecule obtained by replacing 193 rd to 213 th nucleotides from the 5' end of an open reading frame of the NA gene of the influenza virus with the 2S-RBD gene and keeping other nucleotides of the NA gene of the influenza virus unchanged;

the S1-HA gene is a recombinant DNA molecule obtained by inserting an S1 gene between the 51 st nucleotide and the 52 th nucleotide from the 5' end of an open reading frame of the HA gene of the influenza virus and keeping other nucleotides of the HA gene of the influenza virus unchanged;

the S1-NA gene is a recombinant DNA molecule obtained by replacing 193 rd to 213 th nucleotides from the 5' end of an open reading frame of the NA gene of the influenza virus with S1 gene and keeping other nucleotides of the NA gene of the influenza virus unchanged;

the S-RBD gene is a DNA molecule encoding the receptor binding Region (RBD) of the SARS-CoV-2S protein;

the 2S-RBD gene is a DNA molecule formed by connecting two S-RBD genes through 5'-GGTTCAGGCGGATCGGGGAGT-3';

the S-RBD + E + M gene is a DNA molecule formed by connecting a receptor binding Region (RBD) gene for coding SARS-CoV-2S protein and an E protein gene and an M protein gene;

the S1 gene is a DNA molecule encoding the S1 subunit of SARS-CoV-2S protein.

The genes inserted into NS, NA or HA of the present invention include but are not limited to the genes of S protein, S1 protein, S protein RBD region, E protein, M protein, N protein, and other dominant epitopes are also within the scope of the present invention.

In the recombinant virus, the coding amino acid sequence of the S-RBD gene is a protein shown in SEQ ID NO. 1; the S-RBD + E + M gene coding amino acid sequence is protein shown in SEQ ID NO. 3; the amino acid sequence coded by the S1 gene is a protein shown in SEQ ID NO. 11.

In the recombinant virus, the nucleotide sequence of the S-RBD gene is SEQ ID NO. 2; the nucleotide sequence of the S-RBD + E + M gene is SEQ ID NO. 4; the nucleotide sequence of the S1 gene is SEQ ID NO. 12.

In the recombinant virus, the S-RBD + E + M-NS gene is a recombinant DNA molecule obtained by inserting the S-RBD + E + M gene between the 375 nd nucleotide to the 376 nd nucleotide from the 5 ' end of the open reading frame of the NS gene of the influenza virus, linking the 375 th nucleotide from the 5 ' end of the open reading frame of the NS gene of the influenza virus to the S-RBD + E + M gene via 5 ' -TAATG-3 ', linking the S-RBD + E + M gene to the 376 th nucleotide from the 5 ' end of the open reading frame of the NS gene of the influenza virus, and keeping the other nucleotides of the NS gene of the influenza virus unchanged;

the 2S-RBD-NS gene is a recombinant DNA molecule obtained by inserting a 2S-RBD gene between the 375 nd nucleotide from the 5 ' end to the 376 nd nucleotide of the open reading frame of the NS gene of the influenza virus, linking the 375 nd nucleotide from the 5 ' end of the open reading frame of the NS gene of the influenza virus to the 2S-RBD gene through 5 ' -TAATG-3 ', linking the 2S-RBD gene to the 376 nd nucleotide from the 5 ' end of the open reading frame of the NS gene of the influenza virus through 5 ' -TAA-3 ', and keeping other nucleotides of the NS gene of the influenza virus unchanged;

the S-RBD-HA gene is a recombinant DNA molecule obtained by inserting the S-RBD gene between the 51 st nucleotide and the 52 nd nucleotide from the 5 ' end of the open reading frame of the HA gene of the influenza virus, connecting the 51 st nucleotide from the 5 ' end of the open reading frame of the HA gene of the influenza virus and the S-RBD gene through 5'-GCAGCAGCACCTGGAGCAGCA-3', connecting the S-RBD gene with the 52 nd nucleotide from the 5 ' end of the open reading frame of the HA gene of the influenza virus through 5'-GCAGCAGCACCTGGAGCAGCA-3', and keeping other nucleotides of the HA gene of the influenza virus unchanged;

the 2S-RBD-HA gene is a recombinant DNA molecule obtained by inserting a 2S-RBD gene between the 51 st nucleotide and the 52 nd nucleotide from the 5 ' end of an open reading frame of the HA gene of the influenza virus, connecting the 51 st nucleotide from the 5 ' end of the open reading frame of the HA gene of the influenza virus and the 2S-RBD gene through 5'-GCAGCAGCACCTGGAGCAGCA-3', connecting the 2S-RBD gene with the 52 nd nucleotide from the 5 ' end of the open reading frame of the HA gene of the influenza virus through 5'-GCAGCAGCACCTGGAGCAGCA-3', and keeping other nucleotides of the HA gene of the influenza virus unchanged;

the S-RBD-NA gene is a recombinant DNA molecule obtained by replacing 193 rd to 213 th nucleotides from the 5 ' end of an open reading frame of the NA gene of the influenza virus with the S-RBD gene, connecting 192 th nucleotides from the 5 ' end of the open reading frame of the NA gene of the influenza virus with the S-RBD gene, connecting the S-RBD gene with 214 th nucleotides from the 5 ' end of the open reading frame of the NA gene of the influenza virus, and keeping other nucleotides of the NA gene of the influenza virus unchanged;

the 2S-RBD-NA gene is a recombinant DNA molecule obtained by replacing 193 rd to 213 th nucleotides from the 5 ' end of an open reading frame of the NA gene of the influenza virus with the 2S-RBD gene, connecting 192 th nucleotides from the 5 ' end of the open reading frame of the NA gene of the influenza virus with the 2S-RBD gene, connecting the 2S-RBD gene with 214 th nucleotides from the 5 ' end of the open reading frame of the NA gene of the influenza virus, and keeping other nucleotides of the NA gene of the influenza virus unchanged;

the S1-HA gene is a recombinant DNA molecule obtained by inserting an S1 gene between the 51 st nucleotide and the 52 nd nucleotide from the 5 ' end of an open reading frame of the HA gene of the influenza virus, connecting the 51 st nucleotide from the 5 ' end of the open reading frame of the HA gene of the influenza virus with the S1 gene through 5'-GCAGCAGCACCTGGAGCAGCA-3', connecting the S1 gene with the 52 nd nucleotide from the 5 ' end of the open reading frame of the HA gene of the influenza virus through 5'-GCAGCAGCACCTGGAGCAGCA-3', and keeping other nucleotides of the HA gene of the influenza virus unchanged;

the S1-NA gene is a recombinant DNA molecule obtained by replacing 193 rd to 213 th nucleotides from the 5 ' end of an open reading frame of the NA gene of the influenza virus with S1 gene, connecting 192 th nucleotides from the 5 ' end of the open reading frame of the NA gene of the influenza virus to the S1 gene, and connecting the S1 gene to 214 th nucleotides from the 5 ' end of the open reading frame of the NA gene of the influenza virus while keeping the other nucleotides of the NA gene of the influenza virus unchanged.

In the recombinant virus, the nucleotide sequence of the S-RBD + E + M-NS gene is 12 th to 2604 th sites of SEQ ID NO.5 or 12 th to 2605 th sites of SEQ ID NO. 8;

the nucleotide sequence of the S-RBD-HA gene is 12 th to 2593 th positions of SEQ ID NO.7 or 12 th to 2590 th positions of SEQ ID NO. 10;

the nucleotide sequence of the S-RBD-NA gene is 12 th to 2210 th positions of SEQ ID NO.6 or 12 th to 2186 th positions of SEQ ID NO. 9;

the nucleotide sequence of the S1-NA gene is 12 th to 3332 th of SEQ ID NO. 13;

the nucleotide sequence of the S1-HA gene is SEQ ID NO.14, 12 th-3715 th sites;

the nucleotide sequence of the 2S-RBD-NS gene is 12 th to 2451 th of SEQ ID NO. 15;

the nucleotide sequence of the 2S-RBD-NA gene is 12 th to 2990 th of SEQ ID NO. 16;

the nucleotide sequence of the 2S-RBD-HA gene is 12 th to 3373 th of SEQ ID NO. 17.

In the recombinant virus, the host cell is MDCK, Vero and 293T, COS cell or MDCK/293T, MDCK/COS co-cultured cell.

In the invention, the starting plasmid of the recombinant plasmid is a bidirectional transcription expression vector pAD3000 or pHW 2000; when the recombinant plasmids are constructed, each gene is inserted into BsmBI site or Aarl site of bidirectional transcription expression vector pAD3000 or pHW 2000.

In a particular embodiment of the invention, the influenza virus in PB2 of influenza virus, PB1 of influenza virus, PA of influenza virus, NP of influenza virus, M of influenza virus, NS of influenza virus is a cold-adapted, attenuated influenza virus, in particular a cold-adapted, attenuated influenza a virus, more in particular a cold-adapted, attenuated influenza virus strain a/ann arbor/6/60; the influenza virus in HA of the influenza virus and NA of the influenza virus is A type influenza virus, specifically H1N1 subtype influenza virus, and more specifically influenza virus strain A/Michigan/45/2015.

In another specific embodiment of the invention, the influenza virus of PB2 of influenza virus, PB1 of influenza virus, PA of influenza virus, NP of influenza virus, M of influenza virus, NS of influenza virus, HA of influenza virus, NA of influenza virus is influenza a virus, specifically influenza virus of subtype H1N1, more specifically influenza virus strain a/PR/8/1934.

In the recombinant virus, PB2 of the influenza virus is a protein coded by GenBank number AY209938.1(update date is 31-MAY-2005) or GenBank number EF467818.1(update date is 01-MAY-2008); PB1 of the influenza virus is a protein encoded by GenBank number M23972.1(update date of 05-JUN-2006) or GenBank number EF467819.1(update date of 01-MAY-2008); PA of the influenza virus is a protein encoded by GenBank No. M23974.1(update date is 05-JUN-2006) or GenBank No. EF467820.1(update date is 01-MAY-2008); the NP of the influenza virus is protein encoded by GenBank No. M23976.1(update date is 25-MAY-2006) or GenBank No. EF467822.1(update date is 01-MAY-2008); m of the influenza virus is a protein encoded by GenBank with the number of M23978.1(update date of 13-JUL-2006) or the number of EF467824.1(update date of 01-MAY-2008); the NS of the influenza virus is a protein encoded by GenBank No. M23968.1(update date is 05-JUN-2006) or GenBank No. J02150.1(update date is 30-MAY-2006); HA of the influenza virus is a protein encoded by GenBank number KY117023.1 (the update date is VRL 24-AUG-2017) or GenBank number EF467821.1 (the update date is 01-MAY-2008); the NA of the influenza virus is a protein encoded by the GenBank number MK622934.1(update date is 18-MAR-2019) or the GenBank number AF389120.1(update date is 19-SEP-2002).

In the recombinant virus, the GenBank number of the gene of PB2 of the influenza virus is AY209938.1(update date is 31-MAY-2005) or the GenBank number is EF467818.1(update date is 01-MAY-2008); the GenBank number of the gene of PB1 of the influenza virus is M23972.1(update date is 05-JUN-2006) or the GenBank number is EF467819.1(update date is 01-MAY-2008); the GenBank number of the gene of the PA of the influenza virus is M23974.1 (the update date is 05-JUN-2006) or the GenBank number is EF467820.1 (the update date is 01-MAY-2008); the GenBank number of the NP gene of the influenza virus is M23976.1(update date is 25-MAY-2006) or the GenBank number is EF467822.1(update date is 01-MAY-2008); the GenBank number of the M gene of the influenza virus is M23978.1(update date13-JUL-2006) or the GenBank number is EF467824.1(update date 01-MAY-2008); the GenBank number of the gene of NS of the influenza virus is M23968.1(update date is 05-JUN-2006) or the GenBank number is J02150.1(update date is 30-MAY-2006); the GenBank number of the HA gene of the influenza virus is KY117023.1 (the update date is 24-AUG-2017) or the GenBank number is EF467821.1 (the update date is 01-MAY-2008); the GenBank number of the gene of the NA of the influenza virus is MK622934.1(update date is 18-MAR-2019) or the GenBank number is AF389120.1(update date is 19-SEP-2002).

The chimeric vaccine prepared by the recombinant virus is also within the protection scope of the invention.

The invention further provides the application of the recombinant virus and/or the chimeric vaccine in preparing products for preventing and/or treating diseases caused by influenza virus and/or SARS-CoV-2.

The DNA molecule shown in SEQ ID NO.2 or the protein shown in SEQ ID NO.1 is also within the scope of the present invention.

The DNA molecule shown in SEQ ID NO.4 or the protein shown in SEQ ID NO.3 are also within the scope of the present invention.

The DNA molecule shown in SEQ ID NO.12 or the protein shown in SEQ ID NO.11 is also within the scope of the present invention.

The application of the DNA molecule shown in SEQ ID NO.2 or the protein shown in SEQ ID NO.1 in the preparation of products for preventing and/or treating diseases caused by influenza virus and/or SARS-CoV-2 is also within the protection scope of the invention.

The application of the DNA molecule shown in SEQ ID NO.4 or the protein shown in SEQ ID NO.3 in the preparation of products for preventing and/or treating diseases caused by influenza virus and/or SARS-CoV-2 is also within the protection scope of the invention.

The application of the DNA molecule shown in SEQ ID NO.12 or the protein shown in SEQ ID NO.11 in the preparation of products for preventing and/or treating diseases caused by influenza virus and/or SARS-CoV-2 is also within the protection scope of the invention.

The invention operates SARS-CoV-2 epitope and influenza virus genome from gene level, prepares recombinant SARS-CoV-2 vaccine strain with influenza virus as carrier based on RG technology, solves the shortage problem of SARS-CoV-2 vaccine, and is a new milestone in the field of coronavirus vaccine. In addition, the SARS-CoV-2 chimeric vaccine provided by the invention can protect more people from being damaged by influenza virus and SARS-CoV-2, and lays a foundation for realizing the purposes of one-vaccine dual-purpose or one-vaccine multi-purpose.

Drawings

FIG. 1 is a schematic diagram showing the strategy for constructing the S-RBD + E + M-NS gene.

FIG. 2 is a schematic diagram showing the strategy for constructing the S-RBD + E + M-PR8-NS gene.

FIG. 3 is a schematic diagram showing a strategy for constructing the S-RBD-NA gene.

FIG. 4 is a schematic diagram showing a strategy for constructing the S-RBD-PR8-NA gene.

FIG. 5 is a schematic diagram showing a strategy for constructing the S-RBD-HA gene.

FIG. 6 is a schematic diagram showing the strategy for constructing the S-RBD-PR8-HA gene.

FIG. 7 is a schematic diagram showing the construction strategy of the S1-NA gene.

FIG. 8 is a schematic diagram of the construction strategy of the S1-HA gene.

FIG. 9 is a schematic diagram showing a strategy for constructing the 2S-RBD-NS gene.

FIG. 10 is a schematic diagram showing a strategy for constructing the 2S-RBD-NA gene.

FIG. 11 is a schematic diagram showing the strategy for constructing the 2S-RBD-HA gene.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are all conventional ones unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of recombinant novel coronavirus (SARS-CoV-2) vaccine with chimeric S-RBD + E + M-NS epitope

This example provides a recombinant novel coronavirus (SARS-CoV-2), wherein the recombinant SARS-CoV-2 expresses a protein designated as S-RBD + E + M, wherein the S-RBD + E + M is a Receptor Binding Domain (RBD) of SARS-CoV-2S protein and is formed by connecting an E protein and an M protein, the amino acid sequence of the recombinant SARS-CoV-2S protein is represented by SEQ ID NO.3, and the recombinant SARS-CoV-2 is encoded by the S-RBD + E + M gene represented by SEQ ID NO. 4.

Firstly, construction of recombinant plasmids:

1. an NS gene fragment of a cold-adapted attenuated influenza virus strain A/AnnArbor/6/60 is taken as a target point inserted into an S-RBD + E + M gene to construct a recombinant plasmid, which is named pAD-S-RBD + E + M-NS.

Synthesizing the gene shown as SEQ ID NO.5 (the structure is shown in figure 1), wherein, the 1 st site to the 11 th site from the 5 ' end in the SEQ ID NO.5 are protective base + enzyme cutting sites, the 12 th to 40 th sites of the SEQ ID NO.5 are 5 ' end non-coding regions (29nt), the 41 th to 415 th sites of the SEQ ID NO.5 are the first 375 nucleotides of the NS gene of the cold-adapted and attenuated influenza virus strain A/AnnArbor/6/60 (namely, the 1 st to 375 th sites from the 5 ' end of the open reading frame of the NS gene), the 421 st and 2115 th sites of the SEQ ID NO.5 are S-RBD + E + M genes, the 6 th and 2578 th sites of the SEQ ID NO.5 are cold-adapted, 463 nucleotides after the NS gene of the attenuated influenza virus strain A/AnnAlbor/6/60 (namely 376-; the 2605 th to 2617 th positions from the 5 'end in SEQ ID No.5 are 3' end protective base + enzyme cutting sites; the 5 ' end of the S-RBD + E + M gene is connected with 5 ' -TAATG-3 '.

By utilizing a molecular biology method, BsmbI is used for enzyme digestion of a gene sequence shown in SEQ ID NO.5 and a bidirectional transcription expression vector pAD3000 (see a patent: CN200610007909.5 specifically), enzyme digestion fragments are connected, positive cloning is selected, a constructed recombinant plasmid is sequenced and identified, the fragment size is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named pAD-S-RBD + E + M-NS.

The pAD-S-RBD + E + M-NS is a recombinant plasmid obtained by replacing the restriction sites of Bsmbi of pAD3000 with S-RBD + E + M-NS gene segments shown by 12 th to 2604 th sites of SEQ ID NO.5 and keeping other sequences of pAD3000 unchanged.

2. Construction of pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, and pAD-M

The 5 internal viral gene frameworks PB2(GenBank number is AY209938.1(update date is 31-MAY-2005), PB1(GenBank number is M23972.1(update date is 05-JUN-2006)), PA (GenBank number is M23974.1(update date is 05-JUN-2006)), NP (GenBank number is M23976.1(update date is 25-MAY-2006)) and M (GenBank number is M23978.1(update date is 13-JUL-2006)) of the cold-adapted attenuated influenza virus strain A/AnnAdbor/6/60 are respectively connected with the bidirectional transcription expression vector pAD3000 to construct a recombinant plasmid (the specific construction method is shown in patent: CN200610007909.5), and the correctly identified recombinant plasmids are respectively named pAD-PB2, pAD-PB1, pAD-PA, pAD-NP and pAD-M.

The pAD-PB2 is a recombinant plasmid obtained by replacing the restriction enzyme sites of Aarl of pAD3000 with a gene fragment shown in GenBank with AY209938.1 and keeping other sequences of pAD3000 unchanged;

the pAD-PB1 is a recombinant plasmid obtained by replacing the restriction enzyme sites of BmbI of pAD3000 with a gene fragment shown in GenBank with the number M23972.1 and keeping other sequences of pAD3000 unchanged;

the pAD-PA is a recombinant plasmid obtained by replacing enzyme cutting sites of BmbI of pAD3000 with a gene fragment shown in GenBank with the number M23974.1 and keeping other sequences of pAD3000 unchanged;

the pAD-NP is a recombinant plasmid obtained by replacing enzyme cutting sites of BmbI of pAD3000 with a gene fragment shown in GenBank with the number M23976.1 and keeping other sequences of pAD3000 unchanged;

the pAD-M is a recombinant plasmid obtained by replacing the restriction sites of BmbI of pAD3000 with a gene fragment shown in GenBank with the number M23978.1 and keeping other sequences of pAD3000 unchanged.

3. Construction of pAD-HA and pAD-NA

HA (GenBank number KY117023.1 (upper date is 24-AUG-2017)) genes and NA (GenBank number MK622934.1 (upper date is 18-MAR-2019)) genes of influenza virus H1N1 subtype (influenza virus strain A/Michigan/45/2015) are respectively connected with a bidirectional transcription expression vector pAD3000, and the constructed recombinant plasmids (the specific construction method is referred to in the doctor Yangpeng: research on reassortment of H1N1 subtype influenza virus attenuated vaccine strains. doctor paper) are respectively named pAD-HA and pAD-NA after being identified correctly.

The pAD-HA is a recombinant plasmid obtained by replacing enzyme cutting sites of BmbI of pAD3000 with a gene fragment shown in GenBank with a number of KY117023.1 and keeping other sequences of pAD3000 unchanged;

the pAD-NA is a recombinant plasmid obtained by replacing the restriction enzyme sites of BmbI of pAD3000 with a gene fragment shown in GenBank with MK622934.1, and keeping other sequences of pAD3000 unchanged.

Second, rescue of recombinant SARS-CoV-2

1. Cell culture

Uniformly mixing COS-1 cells (ATCC number is CRL-1650TM) with DMEM + 10% FBS culture medium (Sigma) to obtain a COS-1 cell culture system; MDCK cells (ATCC No. CRL-2935TM) were mixed with 1XDMEM + 10% FBS medium (Sigma) to obtain an MDCK cell culture system. Mixing a COS-1 cell culture system and an MDCK cell culture system according to a volume ratio of 2: 1 to obtain COS-1/MDCK co-cultured cells (the number of COS-1 cells is 2 × 10)⁵(ii) a The number of MDCK cells was 1X 10⁵)。

2. Transfection

0.2. mu.g each of 8 plasmids, pAD-S-RBD + E + M-NS, pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-HA and pAD-NA, obtained in the first step were mixed in equal amounts, 10uL transfection reagent (Effectene, purchased from QIAGEN, USA, catalog number: 301425) was added and reacted for 10min at room temperature, COS-1/MDCK CO-cultured cells were CO-transfected at 33 ℃ and 5% CO₂Culturing for 48-60h to obtain cell suspension, inoculating the cell suspension into SPF (specific pathogen free) chick embryos of 9-11 days old, culturing for 72h at 33 ℃, harvesting chick embryo allantoic fluid, and performing a Hemagglutination (HA) test, wherein the HA test result shows that: the average HA titer range of the chick embryo of the recombinant SARS-CoV-2 vaccine strain is 1:2⁷-1:2⁸。

The recombinant SARS-CoV-2 vaccine strain successfully obtaining chimeric S-RBD + E + M-NS epitope is named as rFLU-SARS-CoV-2-NS.

The Hemagglutination (HA) assay specifically comprises the following steps:

(1) a disposable 96-well U-shaped plate is taken, and the name of the sample to be measured is marked on the edge of the plate. (2) The first well of the U-shaped plate was diluted 1:2 to the virus, 100. mu.L of diluted virus mixture was added, and 50. mu.L of virus diluent was added uniformly from the second well.

(3) And repeatedly blowing and beating the first hole by using a 200 mu L pipette to fully and uniformly mix the virus mixed solution, sucking 50 mu L of the mixed solution, adding the sucked mixed solution into the second hole of the U-shaped plate, and diluting the mixed solution in the same way until the mixed solution reaches the penultimate hole. After mixing the penultimate wells, 50 μ L of the virus mixture was discarded, and the last row of wells was reserved as negative control wells.

(4) 50 μ L of 1% suspension of chicken red blood cells was added to each well, mixed well and allowed to stand at room temperature for 30 min.

(5) The observation results are shown as ++++, +++, and + where a layer of red blood cells are uniformly spread on the hole, the edge is irregular basically, the area is +++, the red blood cells form a ring shape, the edge is not smooth, small clumps are arranged on the periphery and are marked as ++, a small cluster is formed at the bottom of the red blood cell hole, the edge is smooth and has a stereoscopic impression, and the red blood cells can be seen to slide like human tears when the plate is inclined for a moment.

(6) Calculation of hemagglutination titer: the result was terminated in + + as an agglutination unit. The highest dilution of virus can cause + + is the erythrocyte agglutination end point, and the reciprocal of the dilution is the erythrocyte agglutination valence, called hemagglutination valence for short.

Thirdly, identification of recombinant SARS-CoV-2

And identifying the rFLU-SARS-CoV-2-NS, observing the virus morphology by an electron microscope, wherein most virus particles are spherical, few virus particles are filamentous, and the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

Inoculating rFLU-SARS-CoV-2-NS to SPF chick embryo of 9-11 days old, taking allantoic fluid of the second generation chick embryo to extract virus RNA, amplifying 8 gene segments through RT-PCR, and sequencing to obtain the result completely consistent with the expected gene sequence. The amplification primers for each of the 8 gene fragments are shown in Table 1.

Amplification primer sequences for the 18 Gene fragments in Table

rFLU-SARS-CoV-2-NS is cultured in chicken embryo in great amount, ultrafiltered, concentrated, purified through sucrose gradient centrifugation, SDS-PAGE electrophoresed, gel stained and decolored to obtain two clear bands of 60KD and 30KD, and this proves the existence of the main components of the antigen.

Fourth, detect Temperature sensitive (ts), Cold adapted (ca) and Attenuated (att) phenotypes of rFLU-SARS-CoV-2-NS

rFLU-SARS-CoV-2-NS is inoculated to MDCK cell, cultured at 25, 33 and 37 deg.c separately, and cell supernatant is collected to determine virus titer. The specific method comprises the following steps:

MDCK cells (ATCC No. CRL-2935TM) were cultured in DMEM (SIGMA) containing 10% FBS (HYCLONE) and seeded in 96-well plates, and after 24 hours, the number of cells reached more than 90%, and the cells were washed 3 times with serum-free DMEM medium. Then diluting 10 times of virus sample rFLU-SARS-CoV-2-NS to infect cells in 96-well plates, 6 wells were set. Cells without viruses are used as negative control, and 6 holes are arranged; 6 wells were prepared with cold-adapted, attenuated influenza virus strain A/AnnArbor/6/60 (described in non-patent document "hop-strong et al, rescue and immune effect evaluation of A/California/07/2009 subtype swine influenza cold-adapted attenuated vaccine strain:. in biochemistry and biophysics progress:. 2010") and A/Michigan/45/2015 (purchased from NIBSC, product number 17-154) as controls. To determine the temperature sensitivity of recombinant rFLU-SARS-CoV-2-NS, the viral plates were charged with CO at temperatures of 33 ℃ and 37 ℃ respectively₂The incubation was carried out in an incubator for 6 days, and the cold acclimatization thereof was measured at 25 ℃ for 10 days. Virus titers were calculated by the Karber method using log10 mean (n-6) TCID₅₀Titer/ml soil SD. The difference between 33-37 ℃ represents a temperature sensitive phenomenon and the difference between 25-33 ℃ represents cold acclimation.

The results are shown in table 2: the rFLU-SARS-CoV-2-NS has both temperature sensitive phenotype and cold adaptation phenotype. The difference in viral titer between 25-33 ℃ was 0.4Log10, indicating that it has a cold-adapted phenotype. The 37 ℃ virus growth titer was less than the 33 ℃ virus titer of 3.9Log 10. The result shows that the recombinant rFLU-SARS-CoV-2-NS is relatively safe, does not propagate in large quantity at the temperature of human body, and can be directly used for the production of vaccines.

TABLE 2 temperature sensitive and Cold adapted phenotype results for rFLU-SARS-CoV-2-NS

Note: ca-ts represents cold-adapted phenotype and temperature-sensitive phenotype

Fifth, in vivo immunization experiment of mice

The rFLU-SARS-CoV-2-NS is ultrafiltered, concentrated, sucrose density gradient centrifuged and purified, and then PBS is used as solvent to prepare attenuated live vaccine. The BALB/c mice of 6-8 weeks old were selected and divided into rFLU-SARS-CoV-2-NS group and PBS group, each group had 20 mice, and 3 replicates were set. rFLU-SARS-CoV-2-NS group: nasal drop immunization of BALB/c mice with live attenuated vaccine for 2 times at 3-week intervals at a dose of 10⁶TCID₅₀Each mouse. PBS group: PBS is replaced by attenuated live vaccine, and the immune mode, immune volume and immune time are consistent with those of the rFLU-SARS-CoV-2-NS group. Collecting serum of mice by eyeground blood collection and centrifugation after primary immunization and secondary immunization of rFLU-SARS-CoV-2-NS group and PBS group for 2 weeks

HI method (reference OIE recommended Standard method) for determining the antibody titer of rFLU-SARS-CoV-2-NS against wild-type influenza A/Michigan/45/2015, NT method (reference: Lokurage Kumari G.Chimeric coronavirus-like antibodies binding polypeptide vaccine and vaccine fragment antigen synthetic vaccine (SCoV) S protein resistant peptide exchange with SCoV [ [ J ]. Pubmed,2008,26(6) ], determination of the antibody titer of rFLU-SARS-CoV-2-NS against SARS-CoV-2S protein (purchased from Kingsry Biotech., product No.: Z03496), results are shown in Table 3, after immunization of SARS-CoV-2-NS animals, antibodies against both the rFLU-SARS-CoV-2S protein and CoV-2S protein can be detected in serum, and after the production of the dual influenza vaccine against the influenza virus A/Michigan/45/2015, the antibody titer against the influenza virus and the influenza vaccine should be indicated And (6) answering.

TABLE 3 detection of the immune Effect of rFLU-SARS-CoV-2-NS

Sixthly, attack toxicity test

Selecting SARS-CoV-2 to infect throat test sample of confirmed clinical patient, inoculating Vero cell (ATCC, CCL81), culturing in DMEM medium (purchased from life, with the product number of C11995500BT), culturing at 35 deg.C for 72 hr, sampling and continuously culturing for five generations to collect virus culture fluid, sampling and using TCLD₅₀Measuring the virus titer to over 7.0, and collecting the virus culture solution containing SARS-CoV-2. The experiment was done in BSL-3 laboratory.

ICR-Tg (hACE2) transgenic mice (purchased from Beijing Huafukang Biotechnology GmbH) with the weight of 13-16 g are selected and randomly divided into 2 groups (namely a PBS control group and an rFLU-SARS-CoV-2-NS group), and 10 mice are selected for each group.

Immunizing a mouse according to the fifth step of 'mouse in vivo immunization experiment',the immunizations were performed 2 times, two weeks apart, and blood samples were collected before immunization, 14 days after the first immunization, and 14 days after the second immunization, respectively. 2 weeks after the second immunization, i.e., PBS control group and rFLU-SARS-CoV-2-NS group were treated with 5X 10 antibody separately⁶PFU dose of SARS-CoV-2 virus culture medium infection.

The results show that: the PBS control group begins to develop symptoms of coronary pneumonia such as listlessness, hair erection, limited activity, small food intake, weight loss and the like 24h after being inoculated with culture solution containing SARS-CoV-2, and the virus titer in lung tissues reaches 10 in the third day after virus challenge^3.5TCID₅₀mL (assay reference: Bao, L., Deng, W., Huang, B.et al.the pathology of SARS-CoV-2 in hACE2 transgenic mice.Nature (2020)); on the third day after challenge, the virus titer in lung tissue was 10 in the mice of the rFLU-SARS-CoV-2-NS group^2.2TCID₅₀The virus titer is obviously reduced compared with the PBS control group. It shows that the rFLU-SARS-CoV-2-NS has certain prevention effect on the novel coronavirus pneumonia.

EXAMPLE 2 preparation of recombinant novel coronavirus (SARS-CoV-2) vaccine of chimeric S-RBD + E + M-PR8-NS

This example provides a recombinant novel coronavirus (SARS-CoV-2), wherein the recombinant SARS-CoV-2 expresses a protein designated as S-RBD + E + M, wherein S-RBD + E + M is a Receptor Binding Domain (RBD) of SARS-CoV-2S protein, the protein E and the protein M are linked, the amino acid sequence of the protein is shown in SEQ ID NO.3, and the protein E is encoded by the gene S-RBD + E + M shown in SEQ ID NO. 4.

Firstly, construction of recombinant plasmids:

1. an NS gene segment (named PR8-NS) of the influenza virus strain A/PR/8/1934 is used as a target point for inserting the epitope gene to construct a recombinant plasmid, and the recombinant plasmid is named pHW-S-RBD + E + M-PR 8-NS.

Synthesizing the gene shown as SEQ ID NO.8 (the structure is shown in FIG. 2), wherein the 1 st to 11 th positions from the 5 ' end in the SEQ ID NO.8 are protective base + enzyme cutting sites, the 12 th to 41 th positions of the SEQ ID NO.8 are 5 ' end non-coding regions (29nt), the 42 th to 416 th positions of the SEQ ID NO.8 are the first 375 nucleotides of the NS gene of the influenza virus strain A/PR/8/1934 (i.e. the 1 st to 375 th positions from the 5 ' end of the open reading frame of the NS gene), the 422 nd and 2116 th positions of the SEQ ID NO.8 are S-RBD + E + M genes, the 2117 nd position 2579 th position of the SEQ ID NO.8 is the last nucleotides of the NS gene of the influenza virus strain A/PR/8/1934 (i.e. the 376 and 812 th positions from the 5 ' end of the open reading frame of the NS gene), the 2580 nd position 2605 nd position of the SEQ ID NO. 2605 nd position is 5 ' end non-coding region (463), the 2606-2618 th site of SEQ ID NO.8 is a protective base plus a restriction enzyme site; the 5 ' end of the S-RBD + E + M gene is connected with 5 ' -TAATG-3 ';

by utilizing a molecular biology method, BmbI cuts a gene sequence shown in SEQ ID NO.8 and a bidirectional transcription expression vector pHW2000 (see a patent: CN200610007909.5 specifically), enzyme cutting fragments are connected, positive clones are selected, the constructed recombinant plasmid is sequenced and identified, the size of the fragment is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named as pHW-S-RBD + E + M-PR 8-NS.

The pHW-PR8-S-RBD + E + M-NS is a recombinant plasmid obtained by replacing the restriction enzyme sites of BmbI of pHW2000 with S-RBD + E + M-PR8-NS gene segments shown by 12-2605 sites of SEQ ID NO.8 and keeping other sequences of pHW2000 unchanged.

2. Construction of pHW-PB2, pHW-PB1, pHW-PA, pHW-NP, pHW-M, pHW-HA, and pHW-NA

The 5 internal viral gene skeletons PB2(GenBank No. EF467818.1(update date 01-MAY-2008)), PB1(GenBank No. EF467819.1(update date 01-MAY-2008)), PA (GenBank No. EF467820.1(update date 01-MAY-2008)), NP (GenBank No. EF467822.1(update date 01-MAY-2008)) and M (GenBank No. EF467824.1(update date 01-MAY-2008)) of the influenza A/PR/8/1934 are respectively connected with a bidirectional transcription expression vector pHW2000 to construct a recombinant plasmid (the specific construction method is shown in patent CN200610007909.5), and the correctly identified recombinant plasmids are respectively named pHW-PB2, pHW-PB1, pHW-PA, pHW-NP and pHW-M.

2 surface genes HA (GenBank number is EF467821.1(update date is 01-MAY-2008)) and NA (GenBank number is AF389120.1(update date is 19-SEP-2002)) of the influenza virus strain A/PR/8/1934 are respectively connected with a bidirectional transcription expression vector pHW2000 to construct recombinant plasmids (the specific construction method is shown in patent: CN200610007909.5), and the correctly identified recombinant plasmids are respectively named as pHW-HA and pHW-NA.

The pHW-PB2 is a recombinant plasmid obtained by replacing the restriction enzyme sites of Aarl of pHW2000 with a gene fragment shown by GenBank under the number EF467818.1 and keeping other sequences of pHW2000 unchanged;

the pHW-PB1 is a recombinant plasmid obtained by replacing enzyme cutting sites of BmbI of pHW2000 with a gene fragment shown in GenBank with the number M23972.1 and keeping other sequences of pHW2000 unchanged;

the pHW-PA is a recombinant plasmid obtained by replacing enzyme cutting sites of BmbI of pHW2000 with a gene fragment shown in GenBank with the number M23974.1 and keeping other sequences of pHW2000 unchanged;

the pHW-NP is a recombinant plasmid obtained by replacing enzyme cutting sites of BmbI of pHW2000 with a gene fragment shown by GenBank with the number M23976.1 and keeping other sequences of pHW2000 unchanged;

the pHW-M is a recombinant plasmid obtained by replacing the restriction enzyme sites of BmbI of pHW2000 with a gene fragment shown by GenBank number M23978.1 and keeping other sequences of pHW2000 unchanged.

The pHW-HA is a recombinant plasmid obtained by replacing enzyme cutting sites of BmbI of pHW2000 with a gene fragment shown in GenBank with a number KY117023.1 and keeping other sequences of pHW2000 unchanged;

the pHW-NA is a recombinant plasmid obtained by replacing the restriction enzyme sites of BmbI of pHW2000 with a gene fragment shown by GenBank number MK622934.1 and keeping other sequences of pHW2000 unchanged.

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of the example 1, and is characterized in that the '8 plasmids of the pAD-S-RBD-NA, the pAD-PB2, the pAD-PB1, the pAD-PA, the pAD-NP, the pAD-M, pAD-NS and the pAD-HA obtained in the first step' are replaced by '8 plasmids of the pHW-S-RBD + E + M-PR8-NS, the pHW-PB2, the pHW-PB1, the pHW-PA, the pHW-NP, the pHW-M, pHW-HA and the pHW-NA obtained in the first step', and the chicken embryo average HA titer of the recombinant SARS-CoV-2 vaccine strain is in the range of 1:2⁷-1:2⁸。

The recombinant SARS-CoV-2 vaccine strain successfully obtaining chimeric S-RBD + E + M-PR8-NS is named as rFLU-SARS-CoV-2-PR 8-NS.

Identification of recombinant SARS-CoV-2

The rFLU-SARS-CoV-2-PR8-NS is identified, the virus morphology is observed by an electron microscope, most virus particles are spherical, a few virus particles are filamentous, the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

rFLU-SARS-CoV-2-PR8-NS is inoculated to SPF chick embryo of 9-11 days old, allantoic fluid of the second generation chick embryo is taken to extract virus RNA, and 8 gene segments are amplified through RT-PCR (the amplification primers of each gene segment are shown in table 1 in example 1) for sequencing, and the result is completely consistent with the expected gene sequence.

The rFLU-SARS-CoV-2-PR8-NS is subjected to mass culture of chick embryos, inactivation, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, two clear bands with the size of about 60KD and 30KD can be observed by the SDS-PAGE, and the existence of main components of the antigen is proved.

Fourth, in vivo immunization experiment of mice

The immunization experiment method is the same as the fifth step in example 1, and is different in that the rFLU-SARS-CoV-2-NS is replaced by the rFLU-SARS-CoV-2-PR8-NS, and the result is shown in the table 4, after the rFLU-SARS-CoV-2-PR8-NS is immunized to animals, the antibody titer aiming at SARS-CoV-2S protein and influenza virus can be detected in the serum, which shows that the vaccine can induce the body to generate double immune response aiming at SARS-CoV-2 and influenza virus after immunization.

TABLE 4 detection of the immune Effect of rFLU-SARS-CoV-2-PR8-NS

Fifth, attack-toxicity test

The immunization and challenge assay procedure was as in step six of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-SARS-CoV-2-PR 8-NS". The results show that: the PBS control group begins to develop symptoms of new coronary pneumonia such as listlessness, hair erection, limited activity, small food intake, weight loss and the like 24h after being inoculated with culture solution containing SARS-CoV-2, and the virus titer in the lung tissue reaches the value after the third day of virus challengeTo 10^3.4TCID₅₀Per mL; on the third day after the challenge of the mice in the rFLU-SARS-CoV-2-PR8-NS group, the virus titer in the lung tissue is 10^2.3TCID₅₀The virus titer is obviously reduced compared with the PBS control group. It shows that the rFLU-SARS-CoV-2-PR8-NS has certain prevention effect on the novel coronavirus pneumonia.

EXAMPLE 3 preparation of recombinant SARS-CoV-2 vaccine of chimeric S-RBD-NA epitope

This example provides a recombinant novel coronavirus (SARS-CoV-2), which recombinant S-RBD-NA expresses a protein designated as S-RBD, which is a Receptor Binding Domain (RBD) of SARS-CoV-2S protein, has an amino acid sequence of SEQ ID No.1, and is encoded by the S-RBD gene shown in SEQ ID No. 2.

Firstly, construction of recombinant plasmids:

1. an NA gene segment of an influenza virus H1N1 subtype (influenza virus strain A/Michigan/45/2015) is used as a target point for inserting an S-RBD gene to construct a recombinant plasmid, and the recombinant plasmid is named as pAD-S-RBD-NA.

Synthesizing the gene shown as SEQ ID NO.6 (the structure is shown in FIG. 3), wherein the 1 st to 11 th sites from the 5 ' end of the SEQ ID NO.6 are protective base + enzyme cutting sites, the 12 th to 34 th sites of the SEQ ID NO.6 are 5 ' end non-coding regions (23nt), the 35 th to 226 th sites of the SEQ ID NO.6 are the first 192 nucleotides of the NA gene of the influenza virus strain A/Michigan/45/2015 (i.e. the 1 st to 192 th sites from the 5 ' end of the open reading frame of the NA gene), the 227 rd and 985 th sites of the SEQ ID NO.6 are S-RBD genes, the 986 rd and 2182 th sites of the SEQ ID NO.6 are the last 7 nucleotides of the NA gene of the influenza virus strain A/Michigan/45/2015 (i.e. the 214 rd and 1410 th sites from the 5 ' end of the open reading frame of the NA gene), the 3 rd and 2210 th sites of the SEQ ID NO.6 are 3 ' end non-coding regions (32), the 2211 th to 2223 th positions from the 5 'end of SEQ ID No.6 are 3' end protective base + enzyme cutting site.

By using a molecular biology method, BmbI cuts the gene shown in SEQ ID NO.6 and a bidirectional transcription expression vector pAD3000 (see a patent: CN200610007909.5 specifically), enzyme cutting fragments are connected, positive clones are selected, the constructed recombinant plasmid is sequenced and identified, the size of the fragment is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named pAD-S-RBD-NA.

The pAD-S-RBD-NA is a recombinant plasmid which is obtained by replacing the restriction enzyme sites of BsmBI of pAD3000 with gene fragments shown in 12 th to 2210 th positions of SEQ ID NO.6 and keeping other sequences of pAD3000 unchanged.

2. Construction of pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS

The internal virus gene skeletons PB2, PB1, PA, NP, M and NS of the cold-adapted attenuated influenza virus strain A/AnnArbor/6/60 are respectively connected with a bidirectional transcription expression vector pAD3000 to construct recombinant plasmids, and the correctly identified recombinant plasmids are respectively named pAD-PB2, pAD-PB1, pAD-PA, pAD-NP and pAD-M, pAD-NS.

The pAD-NS is a recombinant plasmid obtained by replacing the restriction enzyme sites of BmbI of pAD3000 with a gene fragment shown by GenBank number M23968.1(update date is 05-JUN-2006), and keeping other sequences of pAD3000 unchanged;

the other pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, and pAD-M were the same as in example 1.

3、pAD-HA

The HA gene of influenza virus H1N1 subtype (influenza virus strain A/Michigan/45/2015) is connected with a bidirectional transcription expression vector pAD3000 to construct a recombinant plasmid, the constructed recombinant plasmid is named pAD-HA after the correctly identified recombinant plasmid (the same as example 1).

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of the example 1, and is characterized in that the '8 plasmids of the pAD-S-RBD-NA, the pAD-PB2, the pAD-PB1, the pAD-PA, the pAD-NP, the pAD-M, pAD-NS and the pAD-HA obtained in the first step' are replaced by '8 plasmids of the pAD-S-RBD-NA, the pAD-PB2, the pAD-PB1, the pAD-PA, the pAD-NP, the pAD-M, pAD-NS and the pAD-HA obtained in the first step', and the chick embryo average HA titer range of the recombinant SARS-CoV-2 vaccine strain is 1:2⁸-1:2¹⁰。

Successfully obtains the recombinant SARS-CoV-2 vaccine strain of the chimeric S-RBD-NA epitope, which is named as rFLU-S-RBD-NA.

Thirdly, identification of recombinant SARS-CoV-2

And identifying the rFLU-S-RBD-NA, observing the virus morphology by using an electron microscope, wherein most virus particles are spherical, few virus particles are filamentous, and the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

Inoculating the SPF chick embryo of 9-11 days old with the rFLU-S-RBD-NA, taking the allantoic fluid of the second generation chick embryo to extract the virus RNA, amplifying 8 gene segments (the amplification primers of each gene segment are shown in table 1 in example 1) by RT-PCR, and sequencing, wherein the result is completely consistent with the expected gene sequence.

The rFLU-S-RBD-NA is subjected to mass culture of chick embryos, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, and clear two bands with the size of about 60KD and 30KD can be observed on the SDS-PAGE, so that the existence of main components of the antigen is proved.

Fourthly, detecting Temperature sensitive (ts), Cold adapted (ca) and Attenuated (att) phenotype of the rFLU-S-RBD-NA

The rFLU-S-RBD-NA was inoculated into MDCK cells, cultured at 25, 33 and 37 ℃ respectively, and cell supernatants were collected to determine virus titer in the same manner as in example 1, with the results shown in Table 5: the rFLU-S-RBD-NA has both a temperature sensitive phenotype and a cold-adapted phenotype. The difference in viral titer between 25-33 ℃ was at 0.6log10, suggesting that it has a cold-adapted phenotype. The 37 ℃ viral growth titer was less than the 33 ℃ viral titer of 4.1Log 10. The recombinant rFLU-S-RBD-NA is safe, does not propagate in large quantities at the temperature of human body, and can be directly used for vaccine production.

TABLE 5 temperature sensitive and Cold acclimated phenotypic results for rFLU-S-RBD-NA

Fifth, in vivo immunization experiment of mice

The experimental procedure for immunization was the same as in step five of example 1 except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S-RBD-NA", and as a result, as shown in Table 6, the rFLU-S-RBD-NA immunized animals detected antibody titers against SARS-CoV-2S protein and influenza virus in sera after the two-immunization of 1: 1280 and 1: 5120 it shows that the vaccine can induce body to generate dual immune response against SARS-CoV-2 and influenza virus after immunization.

TABLE 6 detection of rFLU-S-RBD-NA immune Effect

Sixthly, attack toxicity test

The immunization and challenge assay procedure was as in step six of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S-RBD-NA". The results show that: the PBS control group begins to develop symptoms of coronary pneumonia such as listlessness, hair erection, limited activity, small food intake, weight loss and the like 24h after being inoculated with culture solution containing SARS-CoV-2, and the virus titer in lung tissues reaches 10 in the third day after virus challenge^3.2TCID₅₀Per mL; on the third day after virus challenge, the virus titer in lung tissue of the mice in the rFLU-S-RBD-NA group was 10^1.9TCID₅₀mL, significantly lower virus titers than PBS control. The results show that the rFLU-S-RBD-NA has certain prevention effect on the novel coronavirus pneumonia.

Experimental example 4 preparation of recombinant novel coronavirus (SARS-CoV-2) vaccine of chimeric S-RBD-PR8-NA

This example provides a recombinant novel coronavirus (SARS-CoV-2), the recombinant S-RBD-PR8-NA expressing a protein named S-RBD, the receptor binding region (RBD region) of the SARS-CoV-2S protein having the amino acid sequence of SEQ ID No.1 encoded by the S-RBD gene shown in SEQ ID No. 2.

Firstly, construction of recombinant plasmids:

1. an NA gene segment (named PR8-NA) of the influenza virus strain A/PR/8/1934 is used as a target point for inserting the epitope gene to construct a recombinant plasmid, and the recombinant plasmid is named pHW-S-RBD-PR 8-NA.

Synthesizing the gene (structure is shown in figure 4) shown as SEQ ID NO.9, wherein, the 1 st to the 11 th sites from the 5 ' end of the SEQ ID NO.9 are protective bases + enzyme cutting sites, the 12 th to the 34 th sites of the SEQ ID NO.9 are 5 ' end non-coding regions (23nt), the 35 th to the 226 th sites of the SEQ ID NO.9 are the first 192 nucleotides of the NA gene of the influenza virus strain A/PR/8/1934, the 227 nd 985 th sites of the SEQ ID NO.9 are S-RBD genes, the 986 nd and 2158 th sites of the SEQ ID NO.9 are the last 1173 nucleotides of the NA gene of the influenza virus strain A/PR/8/1934, the 2159 nd 2186 sites of the SEQ ID NO.9 are 3 ' end non-coding regions (28nt), and the 2187 nd and 2199 nd sites of the SEQ ID NO.9 are protective bases + enzyme cutting sites.

By utilizing a molecular biology method, Bsmbi enzyme cuts the gene shown in SEQ ID NO.9 and a bidirectional transcription expression vector pHW2000 (see a patent: CN200610007909.5 specifically), enzyme cutting fragments are connected, positive cloning is selected, the constructed recombinant plasmid is sequenced and identified, the size of the fragment is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named as pHW-S-RBD-PR 8-NA.

The pHW-S-RBD-PR8-NA is a recombinant plasmid obtained by replacing the restriction enzyme cutting sites of BsmBI of pHW2000 with S-RBD-PR8-NA gene segments shown in 12 th to 2186 th sites of SEQ ID NO.9 and keeping other sequences of pHW2000 unchanged.

2. Construction of pHW-PB2, pHW-PB1, pHW-PA, pHW-NP, and pHW-M, pHW-NS

And respectively connecting the internal virus gene skeletons PB2, PB1, PA, NP, M and NS of the influenza virus strain A/PR/8/1934 with a bidirectional transcription expression vector pHW2000 to construct recombinant plasmids, and respectively naming the correctly identified recombinant plasmids as pHW-PB2, pHW-PB1, pHW-PA, pHW-NP and pHW-M, pHW-NS.

The pHW-NS is a recombinant plasmid obtained by replacing enzyme cutting sites of BsbII of pHW2000 with a gene fragment shown by GenBank number J02150.1(update date is 30-MAY-2006), and keeping other sequences of pHW2000 unchanged;

the other pHW-PB2, pHW-PB1, pHW-PA, pHW-NP, and pHW-M, pHW-HA were as in example 2.

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of example 1, except that the "8 plasmids in total of pAD-S-RBD-NA, pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS and pAD-HA obtained in the first step" are replaced by "8 plasmids in total of pHW-S-RBD-PR8-NA, pHW-PB2, pHW-PB1, pHW-PA, pHW-NP, pHW-M, pHW-NS and pHW-HA obtained in the first step", and the "plasmid" is replaced by the second plasmidThe average HA titer range of the chick embryo of the group SARS-CoV-2 vaccine strain is 1:2⁸-1:2¹⁰。

Successfully obtains the recombinant SARS-CoV-2 vaccine strain of the chimeric S-RBD-PR8-NA, which is named as rFLU-S-RBD-PR 8-NA.

Thirdly, identification of recombinant SARS-CoV-2

The rFLU-S-RBD-PR8-NA is identified, the virus morphology is observed by an electron microscope, most virus particles are spherical, a few virus particles are filamentous, the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

Inoculating the rFLU-S-RBD-PR8-NA into SPF chick embryos of 9-11 days old, taking allantoic fluid of the second generation chick embryos to extract virus RNA, amplifying 8 gene segments (the amplification primers of each gene segment are shown in table 1 in the example) through RT-PCR, and sequencing, wherein the result is completely consistent with the expected gene sequence.

The rFLU-S-RBD-PR8-NA is subjected to mass culture of chick embryos, inactivation, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, two clear bands with the size of about 60KD and 30KD can be observed by the SDS-PAGE, and the existence of main components of the antigen is proved.

Fourth, in vivo immunization experiment of mice

The experimental immunization method was the same as that of step five of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S-RBD-PR 8-NA", and as a result, as shown in Table 7, rFLU-S-RBD-PR8-NA immunized animals and antibody titers against SARS-CoV-2S protein and influenza virus detected in serum after the secondary immunization were 1: 2560 and 1: 5120 it is shown that the vaccine can induce body to generate dual immune response against SARS-CoV-2 and influenza virus after immunization.

TABLE 7 detection of the immune Effect of rFLU-S-RBD-PR8-NA

Fifth, attack-toxicity test

The immunization and challenge assay was performed as in step six of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S-RBD-PR8-NA ". The results show that: the PBS control group begins to develop symptoms of coronary pneumonia such as listlessness, hair erection, limited activity, small food intake, weight loss and the like 24h after being inoculated with culture solution containing SARS-CoV-2, and the virus titer in lung tissues reaches 10 in the third day after virus challenge^3.4TCID₅₀Per mL; on the third day after virus challenge, the virus titer in lung tissue of the mice in the rFLU-S-RBD-PR8-NA group was 10^2.0TCID₅₀The virus titer is obviously reduced compared with the PBS control group. The results show that the rFLU-S-RBD-PR8-NA has certain prevention effect on the novel coronavirus pneumonia.

Experimental example 5: preparation of recombinant novel coronavirus (SARS-CoV-2) vaccine of chimeric S-RBD-HA epitope

This example provides a recombinant novel coronavirus (SARS-CoV-2), the recombinant SARS-CoV-2 expresses a protein designated as S-RBD, which is a receptor binding region (RBD region) of SARS-CoV-2S protein, has an amino acid sequence of SEQ ID No.1, and is encoded by the S-RBD gene shown in SEQ ID No. 2.

Firstly, constructing a recombinant plasmid:

1. an HA gene segment of an influenza virus H1N1 subtype (influenza virus strain A/Michigan/45/2015) is used as a target point for inserting an S-RBD gene, and a recombinant plasmid is constructed by a molecular biological method.

Synthesizing the gene shown as SEQ ID NO.7 (the structure is shown in FIG. 5), wherein the 1 st to 11 th sites from the 5 ' end of the SEQ ID NO.7 are protective base + enzyme cutting sites, the 12 th to 46 th sites of the SEQ ID NO.7 are 3 ' end non-coding regions (35nt), the 47 th to 97 th sites of the SEQ ID NO.7 are signal peptides (51nt) of the HA gene of the influenza virus strain A/Michigan/45/2015 (i.e. the 1 st to 51 th sites from the 5 ' end of the open reading frame of the HA gene), the 119 th and 877 th sites of the SEQ ID NO.7 are S-RBD genes, the 899 nd and 2548 th sites of the SEQ ID NO.7 are the 1650 nucleotides behind the HA gene of the influenza virus strain A/Michigan/45/2015 (i.e. the 52 th to 1701 th sites from the 5 ' end of the open reading frame of the HA gene), the 2549 th site of the SEQ ID NO.7 is 5 ' end non-coding regions (45nt), the 2594 th and 2606 th sites of SEQ ID No.7 are protective bases + enzyme cutting sites, and AAAPGAA polypeptides (gene sequence GCAGCAGCACCTGGAGCAGCA) are added at the 5 'end and the 3' end of the S-RBD gene to serve as a linker.

By utilizing a molecular biology method, BsmBI enzyme cuts the gene shown in SEQ ID NO.7 and a bidirectional transcription expression vector pAD3000 (see the patent: CN200610007909.5 specifically), the enzyme cut fragments are connected, positive clones are selected, the constructed recombinant plasmid is identified by sequencing, the fragment size is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named pAD-S-RBD-HA.

The pAD-S-RBD-HA is a recombinant plasmid obtained by replacing the restriction enzyme sites of BsmBI of pAD3000 with S-RBD-HA gene segments shown in the 12 th to 2593 th sites of SEQ ID NO.7 and keeping other sequences of pAD3000 unchanged.

2. Construction of pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS

6 internal virus gene skeletons PB2, PB1, PA, NP, M and NS of the cold-adapted attenuated influenza virus strain A/AnnArbor/6/60 are respectively connected with a bidirectional transcription expression vector pAD3000 to construct recombinant plasmids, and the correctly identified recombinant plasmids are respectively named pAD-PB2, pAD-PB1, pAD-PA, pAD-NP and pAD-M, pAD-NS.

pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS are as in example 3.

3、pAD-NA

The NA gene of influenza virus H1N1 subtype is connected with a bidirectional transcription expression vector pAD3000 to construct a recombinant plasmid, and the correctly identified recombinant plasmid is named pAD-NA (the same as example 1).

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of the example 1, and is characterized in that the '8 plasmids of the pAD-S-RBD-NA, the pAD-PB2, the pAD-PB1, the pAD-PA, the pAD-NP, the pAD-M, pAD-NS and the pAD-HA obtained in the first step' are replaced by '8 plasmids of the pAD-S-RBD-HA, the pAD-PB2, the pAD-PB1, the pAD-PA, the pAD-NP, the pAD-M, pAD-NS and the pAD-HA obtained in the first step', and the chicken embryo average HA titer range of the recombinant SARS-CoV-2 vaccine strain is 1:2⁷-1:2⁹。

Successfully obtains the recombinant SARS-CoV-2 vaccine strain of the chimeric pAD-S-RBD-HA epitope, which is named as rFLU-S-RBD-HA.

Thirdly, identification of recombinant SARS-CoV-2

And identifying the rFLU-S-RBD-HA, observing the virus morphology by an electron microscope, wherein most virus particles are spherical, few virus particles are filamentous, and the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

Inoculating the rFLU-S-RBD-HA into SPF chick embryos of 9-11 days old, taking allantoic fluid of the second generation chick embryos to extract virus RNA, amplifying 8 gene segments (the amplification primers of each gene segment are shown in the table 1 in the example) through RT-PCR, and sequencing, wherein the result is completely consistent with the expected gene sequence.

The rFLU-S-RBD-HA is subjected to mass culture of chick embryos, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, two clear bands with the sizes of about 60KD and 30KD can be observed, and the existence of main components of the antigen is proved.

Fourthly, detecting Temperature sensitive (ts), Cold adapted (ca) and Attenuated (att) phenotypes of the rFLU-S-RBD-HA

The rFLU-S-RBD-HA was inoculated into MDCK cells, cultured at 25, 33 and 37 ℃ respectively, and cell supernatants were collected to determine virus titer in the same manner as in example 1, with the results shown in Table 8: the rFLU-S-RBD-HA HAs both a temperature sensitive phenotype and a cold-adapted phenotype. The difference in viral titer between 25-33 ℃ was at 0.5log10, indicating that it has a cold-adapted phenotype. The 37 ℃ virus growth titer was less than the 33 ℃ virus titer of 3.7Log 10. The recombinant rFLU-S-RBD-HA is safe, cannot propagate in large quantities at the temperature of a human body, and can be directly used for vaccine production.

TABLE 8 temperature sensitive and Cold acclimation type results for rFLU-S-RBD-HA

Fifth, in vivo immunization experiment in mice

The experimental immunization method was the same as that of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S-RBD-HA", and as a result, as shown in Table 9, the antibody titers against SARS-CoV-2S protein and influenza virus detected in the serum after the secondary immunization were 1: 2560 and 1: 5120 it is shown that the vaccine can induce body to generate dual immune response against SARS-CoV-2 and influenza virus after immunization.

TABLE 9 detection of rFLU-S-RBD-HA immune Effect

Sixth, toxicity counteracting test

The immunization and challenge protocol was the same as step six of example 1 except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S-RBD-HA". The results show that: the PBS control group has symptoms of listlessness, hair erection, limited activity, low food intake, weight loss, etc. after inoculating SARS-CoV-2-containing culture solution for 24h, and virus titer in lung tissue reaches 10 at the third day after virus challenge^3.4TCID₅₀Per mL; on the third day after virus challenge of the mice in the rFLU-S-RBD-HA group, the virus titer in the lung tissue is 10^1.7TCID₅₀mL, significantly lower virus titers than PBS control. The results show that the rFLU-S-RBD-HA HAs certain prevention effect on the novel coronavirus pneumonia.

Experimental example 6 preparation of recombinant SARS-CoV-2 vaccine chimeric S-RBD-PR8-HA

This example provides a recombinant novel coronavirus (SARS-CoV-2), the recombinant SARS-CoV-2 expressing a protein designated as S-RBD, the receptor binding region (RBD region) of the SARS-CoV-2S protein having the amino acid sequence of SEQ ID No.1 encoded by the S-RBD gene shown in SEQ ID No. 2.

Firstly, construction of recombinant plasmids:

1. an HA gene segment (named as PR8-HA) of an influenza virus H1N1 subtype (influenza virus strain A/PR/8/1934) is used as a target point for inserting the epitope gene to construct a recombinant plasmid, and the recombinant plasmid is named as pHW-S-RBD-PR 8-HA.

Synthesizing the gene (structure is shown in figure 6) shown as SEQ ID NO.10, wherein the 1 st to 11 th sites of the SEQ ID NO.10 from the 5 ' end are protective base + enzyme cutting sites, the 12 th to 46 th sites of the SEQ ID NO.10 are 3 ' end non-coding regions (35nt), the 47 th to 97 th sites of the SEQ ID NO.10 are signal peptides (51nt) of HA genes of influenza virus strains A/PR/8/1934, the 119 th and 877 th sites of the SEQ ID NO.10 are S-RBD genes, the 899 th and 2545 th sites of the SEQ ID NO.10 are the last 1647 nucleotides of the HA genes of the influenza virus strains A/PR/8/1934, the 2546 th and 2590 th sites of the SEQ ID NO.10 are 5 ' end non-coding regions (45nt), the 2591 th and 2603 rd sites of the SEQ ID NO.10 are protective base + enzyme cutting sites, and the 5 ' end and the 3 ' end of the S-RBD genes are connected with an APGAA polypeptide (AAGAA GCAGCAGCACCTGGAGCAGCA) as a linker gene.

By utilizing a molecular biology method, BsmBI enzyme cuts the gene shown in SEQ ID NO.10 and a bidirectional transcription expression vector pHW2000 (see a patent: CN200610007909.5 specifically), enzyme cut fragments are connected, positive clones are selected, the constructed recombinant plasmid is sequenced and identified, the size of the fragment is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named as pHW-S-RBD-PR 8-HA.

The pHW-S-RBD-PR8-HA is a recombinant plasmid obtained by replacing the restriction enzyme cutting sites of BsmBI of pHW2000 with S-RBD-PR8-HA gene segments shown in 12 th to 2590 th sites of SEQ ID NO.10 and keeping other sequences of pHW2000 unchanged.

2. Construction of pHW-PB2, pHW-PB1, pHW-PA, pHW-NP, and pHW-M, pHW-NS

And 6 internal virus gene skeletons PB2, PB1, PA, NP, M and NS of the influenza virus strain A/PR/8/1934 are respectively connected with a bidirectional transcription expression vector pHW2000 to construct recombinant plasmids, and the correctly identified recombinant plasmids are respectively named as pHW-PB2, pHW-PB1, pHW-PA, pHW-NP and pHW-M, pHW-NS.

pHW-PB2, pHW-PB1, pHW-PA, pHW-NP, and pHW-M, pHW-NA were as in example 2; pHW-NS is the same as in example 4.

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of example 1, and is characterized in that the '8 plasmids in total of pAD-S-RBD-NA, pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS and pAD-HA obtained in the first step' are replaced by '8 plasmids in total of pHW-S-RBD-PR8-HA, pHW-PB2, pHW-PB1, pHW-PA, pHW-NP, pHW-M, pHW-NS and pHW-HA obtained in the first step', and the recombinant SARS-The average HA titer range of chick embryo of the CoV-2 vaccine strain is 1:2⁷-1:2⁹。

Successfully obtains the recombinant SARS-CoV-2 vaccine strain of the chimeric pHW-S-RBD-PR8-HA, which is named as rFLU-S-RBD-PR 8-HA.

Identification of recombinant SARS-CoV-2

The rFLU-S-RBD-PR8-HA is identified, the virus morphology is observed by an electron microscope, most virus particles are spherical, a few virus particles are filamentous, the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

Inoculating the rFLU-S-RBD-PR8-HA into SPF chick embryos of 9-11 days old, taking allantoic fluid of the second generation chick embryos to extract virus RNA, amplifying 8 gene segments (the amplification primers of each gene segment are shown in table 1 in example 1) through RT-PCR, and sequencing, wherein the result is completely consistent with the expected gene sequence.

The rFLU-S-RBD-PR8-HA is subjected to mass culture of chick embryos, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, two clear bands with the sizes of about 60KD and 30KD can be observed, and the existence of main components of the antigen is proved.

Fourth, in vivo immunization experiment of mice

The experimental immunization method was the same as that of step five of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S-RBD-PR 8-HA", and as a result, as shown in Table 10, rFLU-S-RBD-PR 8-HA-immunized animals had antibody titers against SARS-CoV-2S protein and influenza virus detected in serum after the secondary immunization of 1: 1280 and 1: 5120 it is shown that the vaccine can induce body to generate dual immune response against SARS-CoV-2 and influenza virus after immunization.

TABLE 10 detection of the immune Effect of rFLU-S-RBD-PR8-HA

Fifth, attack-toxicity test

The immunization and challenge assay procedure was as in step six of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S-RBD-PR 8-HA".The results show that: the PBS control group has symptoms of listlessness, hair erection, limited activity, low food intake, weight loss, etc. after inoculating SARS-CoV-2-containing culture solution for 24h, and virus titer in lung tissue reaches 10 at the third day after virus challenge^3.5TCID₅₀Per mL; on the third day after virus challenge of the mice in the rFLU-S-RBD-PR8-HA group, the virus titer in lung tissues is 10^1.5TCID₅₀The virus titer is obviously reduced compared with the PBS control group. The results show that the rFLU-S-RBD-PR8-HA HAs certain prevention effect on the novel coronavirus pneumonia.

Experimental example 7 preparation of recombinant SARS-CoV-2 vaccine chimeric S1-NA epitope

This example provides a recombinant novel coronavirus (SARS-CoV-2), the recombinant S1-NA expresses the protein named S1, and the S1 is the S1 subunit of SARS-CoV-2S protein, the amino acid sequence of the subunit is SEQ ID NO.11, and the subunit is encoded by the S-RBD gene shown in SEQ ID NO. 12.

Firstly, construction of recombinant plasmids:

1. an NA gene segment of an influenza virus H1N1 subtype (influenza virus strain A/Michigan/45/2015) is used as a target point inserted into an S1 gene to construct a recombinant plasmid which is named pAD-S1-NA.

Synthesizing the gene shown as SEQ ID NO.13 (the structure is shown in FIG. 7), wherein, the 1 st to 11 th sites from the 5 ' end of the SEQ ID NO.13 are protective base + enzyme cutting sites, the 12 th to 34 th sites of the SEQ ID NO.13 are 5 ' end non-coding regions (23nt), the 35 th to 226 th sites of the SEQ ID NO.13 are the first 192 nucleotides of the NA gene of the influenza virus strain A/Michigan/45/2015 (i.e. the 1 st to 192 th sites from the 5 ' end of the open reading frame of the NA gene), the 227 rd and 2107 th sites of the SEQ ID NO.13 are S-RBD genes, the 2108 nd and 3304 th sites of the SEQ ID NO.13 are the last 1197 nucleotides of the NA gene of the influenza virus strain A/Michigan/45/2015 (i.e. the 214 nd and 1410 th sites from the 5 ' end of the open reading frame of the NA gene), the 3305 rd site of the SEQ ID NO.13 is 3 ' end non-coding region (28nt), the 3333 th to 3345 th positions of SEQ ID No.13 from the 5 'end are protective bases + enzyme cutting sites of the 3' end.

By utilizing a molecular biology method, Bsmbi enzyme cuts the gene shown in SEQ ID NO.13 and a bidirectional transcription expression vector pAD3000 (see the patent: CN200610007909.5 specifically), enzyme cutting fragments are connected, positive cloning is selected, the constructed recombinant plasmid is identified by sequencing, the fragment size is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named pAD-S1-NA.

The pAD-S1-NA is a recombinant plasmid which is obtained by replacing the restriction sites of BsmBI of pAD3000 with gene fragments shown in 12 th-3332 th sites of SEQ ID NO.13 and keeping other sequences of pAD3000 unchanged.

2. Construction of pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS

pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS are as in example 3.

3、pAD-HA

The HA gene of influenza virus H1N1 subtype (influenza virus strain A/Michigan/45/2015) is connected with a bidirectional transcription expression vector pAD3000 to construct a recombinant plasmid, the constructed recombinant plasmid is named pAD-HA after the correctly identified recombinant plasmid (the same as the example 1).

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of example 1, except that the 8 plasmids of pAD-S-RBD-NA, pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS and pAD-HA obtained in the first step are replaced by the 8 plasmids of pAD-S1-NA, pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS and pAD-HA obtained in the first step, and finally the recombinant SARS-CoV-2 vaccine strain of the chimeric S1-NA epitope is successfully obtained and named as rFLU-S1-NA.

Identification of recombinant SARS-CoV-2

And identifying the rFLU-S1-NA, observing the virus morphology by an electron microscope, wherein most of virus particles are spherical, few virus particles are filamentous, and the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

Inoculating the SPF chick embryo of 9-11 days old with the rFLU-S1-NA, taking the allantoic fluid of the second generation chick embryo to extract the virus RNA, amplifying 8 gene segments (the amplification primers of each gene segment are shown in the table 1 in the example) by RT-PCR, and sequencing, wherein the result is completely consistent with the expected gene sequence.

The rFLU-S1-NA is subjected to mass culture of chick embryos, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, and two clear bands with the sizes of about 60KD and 30KD can be observed, thereby proving that the main components of the antigen exist.

Fourthly, detecting that the rFLU-S1-NA has Temperature sensitive (ts), Cold adapted (ca) and Attenuated (att) phenotypes, and the specific method is the same as that of the example 1.

Fifth, in vivo immunization experiment of mice

The experimental immunization method was the same as that of step five of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S1-NA", and the results are shown in Table 11, in which rFLU-S1-NA-immunized animals and antibody titers against SARS-CoV-2S protein and influenza virus detected in serum after the secondary immunization were 1: 2650 and 1: 5120 it is shown that the vaccine can induce body to generate dual immune response against SARS-CoV-2 and influenza virus after immunization.

TABLE 11 detection of the immune Effect of rFLU-S1-NA

Sixthly, attack toxicity test

The immunization and challenge assay procedure was as in step six of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S1-NA". The results show that: the PBS control group begins to develop symptoms of coronary pneumonia such as listlessness, hair erection, limited activity, small food intake, weight loss and the like 24h after being inoculated with culture solution containing SARS-CoV-2, and the virus titer in lung tissues reaches 10 in the third day after virus challenge^3.3TCID₅₀Per mL; on the third day after virus challenge, the virus titer in lung tissue of the mice in the rFLU-S1-NA group was 10^1.2TCID₅₀The virus titer is obviously reduced compared with the PBS control group. Say thatThe Ming rFLU-S1-NA has certain prevention effect on the novel coronavirus pneumonia.

Experimental example 8: preparation of recombinant novel coronavirus (SARS-CoV-2) vaccine of chimeric S1-HA epitope

This example provides a recombinant novel coronavirus (SARS-CoV-2), the recombinant SARS-CoV-2 expressing the protein designated S1, the S1 being the S1 subunit of the SARS-CoV-2S protein. The amino acid sequence of the polypeptide is SEQ ID NO.11 and is coded by an S1 gene shown in SEQ ID NO. 12.

Firstly, construction of recombinant plasmids:

1. an HA gene segment of an influenza virus H1N1 subtype (influenza virus strain A/Michigan/45/2015) is taken as a target point inserted into an S1 gene, and a recombinant plasmid is constructed by a molecular biological method.

Synthesizing the gene shown as SEQ ID NO.14 (structure shown in FIG. 8), wherein the 1 st to 11 th positions from the 5 ' end of the SEQ ID NO.14 are protective base + enzyme cutting sites, the 12 th to 46 th positions of the SEQ ID NO.14 are 3 ' end non-coding regions (35nt), the 47 th to 97 th positions of the SEQ ID NO.14 are signal peptides (51nt) of the HA gene of the influenza virus strain A/Michigan/45/2015 (i.e. the 1 st to 51 th positions from the 5 ' end of the open reading frame of the HA gene), the 119 th and 1999 th positions of the SEQ ID NO.14 are S1 genes, the 2021 nd and 3670 th positions of the SEQ ID NO.14 are the last 1650 nucleotides of the HA gene of the influenza virus strain A/Michigan/45/2015 (i.e. the 52 nd to 1701 from the 5 ' end of the open reading frame of the HA gene), the 3671 th position of the SEQ ID NO.14 is 5 ' end non-coding region (45), the 3716-3728 th site of SEQ ID No.14 is protective base + enzyme cutting site, and AAAPGAA polypeptide (gene sequence GCAGCAGCACCTGGAGCAGCA) is added at the 5 'end and the 3' end of the S1 gene and is used as a linker to be connected with the HA gene.

By using a molecular biology method, BsmBI enzyme cuts the gene shown in SEQ ID NO.13 and a bidirectional transcription expression vector pAD3000 (see a patent: CN200610007909.5 specifically), enzyme cut fragments are connected, positive clones are selected, the constructed recombinant plasmid is sequenced and identified, the size of the fragment is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named pAD-S1-HA.

The pAD-S1-HA is a recombinant plasmid obtained by replacing the restriction enzyme sites of BsmBI of pAD3000 with S1-HA gene fragments shown in 12 th to 3715 th sites of SEQ ID NO.14 and keeping other sequences of pAD3000 unchanged.

2. Construction of pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS

pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS are as in example 1.

3、pAD-NA

The NA gene of influenza virus H1N1 subtype is connected with a bidirectional transcription expression vector pAD3000 to construct a recombinant plasmid, the constructed recombinant plasmid is named pAD-NA by the correctly identified recombinant plasmid (the same as example 1).

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of example 1, and is different from the method in that the '8 plasmids of the pAD-S-RBD-NA, the pAD-PB2, the pAD-PB1, the pAD-PA, the pAD-NP, the pAD-M, pAD-NS and the pAD-HA which are obtained in the first step' are replaced by '8 plasmids of the pAD-S1-HA, the pAD-PB2, the pAD-PB1, the pAD-PA, the pAD-NP, the pAD-M, pAD-NS and the pAD-NA which are obtained in the first step', and finally the recombinant SARS-CoV-2 vaccine strain of the chimeric S1-NA epitope is successfully obtained and is named as rFLU-S1-HA.

Identification of recombinant SARS-CoV-2

And identifying the rFLU-S1-HA, observing the virus morphology by an electron microscope, wherein most of virus particles are spherical, few virus particles are filamentous, and the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

Inoculating the 9-11-day-old SPF chick embryos with the rFLU-S1-HA, taking the allantoic fluid of the second generation chick embryos to extract virus RNA, amplifying 8 gene segments (the amplification primers of each gene segment are shown in the table 1 in the example) through RT-PCR, and sequencing, wherein the result is completely consistent with the expected gene sequence.

The rFLU-S1-HA is subjected to mass culture of chick embryos, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, two clear bands with the size of about 60KD and 30KD can be observed, and the existence of main components of the antigen is proved.

Fourthly, detecting that the rFLU-S1-HA HAs Temperature sensitive (ts), Cold adapted (ca) and Attenuated (att) phenotypes, and the specific method is the same as that of the example 1.

Fifth, in vivo immunization experiment of mice

The experimental immunization method was the same as that of step five of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S1-HA", and the results are shown in Table 12, wherein the antibody titers against SARS-CoV-2S protein and influenza virus detected in the serum after the immunization of animals with rFLU-S1-HA were 1: 2560 and 1: 5120 it shows that the vaccine can induce body to generate dual immune response against SARS-CoV-2 and influenza virus after immunization.

TABLE 12 detection of the immune Effect of rFLU-S1-HA

Sixthly, attack toxicity test

The immunization and challenge protocol was the same as step six of example 1 except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-S1-HA". The results show that: the PBS control group begins to develop symptoms of coronary pneumonia such as listlessness, hair erection, limited activity, small food intake, weight loss and the like 24h after being inoculated with culture solution containing SARS-CoV-2, and the virus titer in lung tissues reaches 10 in the third day after virus challenge^3.1TCID₅₀Per mL; on the third day after challenge, the virus titer in lung tissues was 10 in the mice of the rFLU-S1-HA group^1.4TCID₅₀The virus titer is obviously reduced compared with the PBS control group. The results show that the rFLU-S1-HA HAs certain prevention effect on the novel coronavirus pneumonia.

EXAMPLE 9 preparation of recombinant novel coronavirus (SARS-CoV-2) vaccine with chimeric 2S-RBD-NS epitope

This example provides a recombinant novel coronavirus (SARS-CoV-2), wherein the recombinant SARS-CoV-2 expresses a protein named 2S-RBD, the 2S-RBD is a Receptor Binding Domain (RBD) of SARS-CoV-2S protein, the two RBDs are connected by a linker (GGTTCAGGCGGATCGGAGT), and the amino acid sequence of the S-RBD is shown in SEQ ID NO.1 and is encoded by the S-RBD gene shown in SEQ ID NO. 2.

Firstly, construction of recombinant plasmids:

1. an NS gene fragment of a cold-adapted attenuated influenza virus strain A/AnnArbor/6/60 is taken as a target point for inserting 2 repeated S-RBD genes to construct a recombinant plasmid which is named pAD-2S-RBD-NS.

Synthesizing the gene (structure is shown in figure 9) shown as SEQ ID NO.15, wherein, the 1 st to 11 th from the 5 ' end in the SEQ ID NO.15 are protective base + enzyme cutting sites, the 12 th to 40 th positions of the SEQ ID NO.15 are 5 ' end non-coding regions (29nt), the 41 th to 415 th positions of the SEQ ID NO.15 are the first 375 nucleotides of the NS gene of the cold-adapted and attenuated influenza virus strain A/AnnArbor/6/60 (namely, the 1 st to 375 th positions from the 5 ' end of the open reading frame of the NS gene), the 421 nd and 1959 th positions of the SEQ ID NO.15 are 2S-RBD genes, the 1963 nd and 2425 th positions of the SEQ ID NO.15 are cold-adapted, 463 nucleotides after the NS gene of the attenuated influenza virus strain A/AnnAlbor/6/60 (namely 376-; the 2452 nd site to the 2464 th site from the 5 'end in SEQ ID No.15 are a 3' end protective base + enzyme cutting site; the 5 'end of the 2S-RBD gene is connected with 5' -TAATG-3 ', and the 3' end is connected with 5 '-TAA-3'.

By utilizing a molecular biology method, BsmbI is used for enzyme digestion of a gene sequence shown in SEQ ID NO.15 and a bidirectional transcription expression vector pAD3000 (see a patent: CN200610007909.5 specifically), enzyme digestion fragments are connected, positive cloning is selected, a constructed recombinant plasmid is sequenced and identified, the fragment size is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named pAD-2S-RBD-NS.

The pAD-2S-RBD-NS is a recombinant plasmid obtained by replacing the restriction enzyme sites of BsbII of pAD3000 with 2S-RBD-NS gene segments shown by 12 th to 2451 th sites of SEQ ID NO.15 and keeping other sequences of pAD3000 unchanged.

2. The construction methods of pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-HA and pAD-NA are the same as in example 1.

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of the example 1, and is characterized in that the ' 8 plasmids of the pAD-2S-RBD-NS, the pAD-PB2, the pAD-PB1, the pAD-PA, the pAD-NP, the pAD-M, pAD-HA and the pAD-NA obtained in the first step are replaced by ' 8 plasmids of the pHW-S-RBD-PR8-HA, the pHW-PB2, the pHW-PB1, the pHW-PA, the pHW-NP, the pHW-M, pHW-NS and the pHW-HA obtained in the first step ', and the recombinant SARS-CoV-2 vaccine strain successfully obtaining the chimeric 2S-RBD-NS epitope is named as rFLU-2S-RBD-NS.

Thirdly, identification of recombinant SARS-CoV-2

And identifying the rFLU-2S-RBD-NS, observing the virus morphology by an electron microscope, wherein most virus particles are spherical, few virus particles are filamentous, and the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

Inoculating the rFLU-2S-RBD-NS to SPF chick embryos of 9-11 days old, taking allantoic fluid of the second generation chick embryos to extract virus RNA, amplifying 8 gene segments through RT-PCR and sequencing, wherein the result is completely consistent with the expected gene sequence. The amplification primers for each of the 8 gene fragments are shown in Table 1.

The rFLU-2S-RBD-NS is subjected to mass culture of chick embryos, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, and clear two bands with the size of about 60KD and 30KD can be observed on the SDS-PAGE, thereby proving that the main components of the antigen exist.

Fourthly, detecting that the rFLU-2S-RBD-NS has Temperature sensitive (ts), Cold adapted (ca) and Attenuated (att) phenotypes, and the specific method is the same as that of the example 1.

Fifth, in vivo immunization experiment of mice

The experimental immunization method was the same as that of step five of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-2S-RBD-NS", and the results are shown in Table 13, in which rFLU-2S-RBD-NS-immunized animals and serum after the secondary immunization detected antibody titers against SARS-CoV-2S protein and influenza virus were 1: 3200 and 1: 5120 it shows that the vaccine can induce body to generate dual immune response against SARS-CoV-2 and influenza virus after immunization.

TABLE 13 detection of rFLU-2S-RBD-NS immune Effect

Sixthly, attack toxicity test

The immunization and challenge assay procedure was as in step six of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-2S-RBD-NS". The results show that: the PBS control group has symptoms of listlessness, hair erection, limited activity, low food intake, weight loss, etc. after inoculating SARS-CoV-2-containing culture solution for 24h, and virus titer in lung tissue reaches 10 at the third day after virus challenge^3.4TCID₅₀Per mL; on the third day after the mice in the rFLU-2S-RBD-NS group are attacked, the virus titer in the lung tissue is not detected and is obviously reduced compared with that in a PBS control group. The rFLU-2S-RBD-NS has certain prevention effect on the novel coronavirus pneumonia.

EXAMPLE 10 preparation of recombinant SARS-CoV-2 vaccine comprising chimeric 2S-RBD-NA epitope

This example provides a recombinant novel coronavirus (SARS-CoV-2), wherein the recombinant SARS-CoV-2 expresses a protein named 2S-RBD, the 2S-RBD is a Receptor Binding Domain (RBD) of SARS-CoV-2S protein, the two RBDs are repeated twice and are connected by a linker (GGTTCAGGCGGATCGGAGT), the amino acid sequence of the S-RBD is shown in SEQ ID NO.1 and is encoded by the S-RBD gene shown in SEQ ID NO. 2.

Firstly, construction of recombinant plasmids:

1. an NA gene segment of an influenza virus H1N1 subtype (influenza virus strain A/Michigan/45/2015) is used as a target point for inserting a 2S-RBD gene to construct a recombinant plasmid which is named pAD-2S-RBD-NA.

Synthesizing the gene (structure is shown in FIG. 10) shown as SEQ ID NO.16, wherein, the 1 st to 11 th sites from the 5 ' end of the SEQ ID NO.16 are protective base + enzyme cutting sites, the 12 th to 34 th sites of the SEQ ID NO.16 are 5 ' end non-coding regions (23nt), the 35 th to 226 th sites of the SEQ ID NO.16 are the first 192 nucleotides of the NA gene of the influenza strain A/Michigan/45/2015 (i.e. the 1 st to 192 th sites from the 5 ' end of the open reading frame of the NA gene), the 227 rd and 1765 th sites of the SEQ ID NO.16 are 2S-RBD genes, the 1766 nd and 2962 th sites of the SEQ ID NO.16 are the last 1197 nucleotides of the NA gene of the influenza strain A/Michigan/45/2015 (i.e. the 214 nd sites from the 5 ' end and 1410 th sites of the open reading frame of the NA gene), the 2963 nd site of the SEQ ID NO.16 is the 293 ' end non-coding region (32nt), the 2991 th to 3003 th positions of SEQ ID No.16 from the 5 'end are 3' end protective base + enzyme cutting site.

By using a molecular biology method, BmbI cuts the gene shown in SEQ ID NO.16 and a bidirectional transcription expression vector pAD3000 (see a patent: CN200610007909.5 specifically), enzyme cutting fragments are connected, positive clones are selected, the constructed recombinant plasmid is sequenced and identified, the size of the fragment is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named pAD-2S-RBD-NA.

The pAD-2S-RBD-NA is a recombinant plasmid obtained by replacing the restriction enzyme sites of BsmBI of pAD3000 with 2S-RBD-NA gene segments shown in 12 th to 2990 th positions of SEQ ID NO.16 and keeping other sequences of pAD3000 unchanged.

2. pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS, and pAD-HA were constructed as in example 1.

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of example 3, except that pAD-S-RBD-NA is replaced by pAD-2S-RBD-NA, and the average HA titer range of the chick embryo of the recombinant SARS-CoV-2 vaccine strain is 1:2⁸-1:2⁹。

Successfully obtains the recombinant SARS-CoV-2 vaccine strain of the chimeric 2S-RBD-NA epitope, which is named as rFLU-2S-RBD-NA.

Identification of recombinant SARS-CoV-2

And identifying the rFLU-2S-RBD-NA, observing the virus morphology by using an electron microscope, wherein most virus particles are spherical, few virus particles are filamentous, the sizes of the virus particles are not completely consistent, the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses.

Inoculating the SPF chick embryo of 9-11 days old with the rFLU-2S-RBD-NA, taking the allantoic fluid of the second generation chick embryo to extract the virus RNA, amplifying 8 gene segments (the amplification primers of each gene segment are shown in table 1 in example 1) by RT-PCR, and sequencing, wherein the result is completely consistent with the expected gene sequence.

The rFLU-2S-RBD-NA is subjected to mass culture of chick embryos, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, and clear two bands with the size of about 60KD and 30KD can be observed on the SDS-PAGE, thereby proving that the main components of the antigen exist.

Fourthly, detecting that the rFLU-2S-RBD-NA has Temperature sensitive (ts), Cold adapted (ca) and Attenuated (att) phenotypes, and the specific method is the same as that of the example 1.

Fifth, in vivo immunization experiment of mice

The experimental immunization method was the same as that of step five of example 1 except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-2S-RBD-NA", and the results are shown in Table 14: when the rFLU-2S-RBD-NA immune animals are immunized, the antibody titer aiming at SARS-CoV-2S protein and influenza virus is respectively detected in serum after the secondary immunization, the antibody titer is 1: 3840 and 1: 5120 it is shown that the vaccine can induce body to generate dual immune response against SARS-CoV-2 and influenza virus after immunization.

TABLE 14 detection of rFLU-2S-RBD-NA immune Effect

Sixthly, attack toxicity test

The immunization and challenge protocol was the same as step six of example 1 except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-2S-RBD-NA". The results show that: the PBS control group begins to develop symptoms of coronary pneumonia such as listlessness, hair erection, limited activity, small food intake, weight loss and the like 24h after being inoculated with culture solution containing SARS-CoV-2, and the virus titer in lung tissues reaches 10 in the third day after virus challenge^3.1TCID₅₀Per mL; on the third day after the mice in the rFLU-2S-RBD-NA group are attacked, the virus titer in the lung tissue is not detected and is obviously reduced compared with that in the PBS control group. The rFLU-2S-RBD-NA has certain prevention effect on the novel coronavirus pneumonia.

Experimental example 11: preparation of recombinant novel coronavirus (SARS-CoV-2) vaccine of chimeric 2S-RBD-HA epitope

This example provides a recombinant novel coronavirus (SARS-CoV-2), the recombinant SARS-CoV-2 expresses a protein named 2S-RBD, the 2S-RBD is a Receptor Binding Domain (RBD) of SARS-CoV-2S protein repeated twice and linked by a linker (GGTTCAGGCGGATCGGGGAGT), the amino acid sequence of S-RBD is SEQ ID NO.1 and is encoded by the S-RBD gene shown in SEQ ID NO. 2.

Firstly, construction of recombinant plasmids:

Synthesizing the gene shown as SEQ ID NO.17 (structure shown in FIG. 11), wherein the 1 st to 11 th positions from the 5 ' end of the SEQ ID NO.17 are protective base + enzyme cutting sites, the 12 th to 46 th positions of the SEQ ID NO.17 are 3 ' end non-coding regions (35nt), the 47 th to 97 th positions of the SEQ ID NO.17 are signal peptides (51nt) of the HA gene of the influenza virus strain A/Michigan/45/2015 (i.e., the 1 st to 51 th positions from the 5 ' end of the open reading frame of the HA gene), the 119 th position and 1657 th positions of the SEQ ID NO.17 are 2S-RBD genes, the 1679 th and 3328 th positions of the SEQ ID NO.17 are the latter 1650 nucleotides of the HA gene of the influenza virus strain A/Michigan/45/2015 (i.e., the 52 th to 1701 th positions from the 5 ' end of the open reading frame of the HA gene), and the 3329 th position and 3373 nd positions of the SEQ ID NO.17 are 5 ' end non-coding regions (45nt), the 3374-3386 site of SEQ ID No.17 is protective base + enzyme cutting site, and AAAPGAA polypeptide (gene sequence GCAGCAGCACCTGGAGCAGCA) is added at both the 5 'end and the 3' end of the S-RBD gene to be connected with HA gene as a linker.

By using a molecular biology method, BsmBI enzyme cuts the gene shown in SEQ ID NO.17 and a bidirectional transcription expression vector pAD3000 (see a patent: CN200610007909.5 specifically), enzyme cut fragments are connected, positive clones are selected, the constructed recombinant plasmid is sequenced and identified, the size of the fragment is completely consistent with the expected size, no gene mutation exists, and the correctly identified recombinant plasmid is named pAD-2S-RBD-HA.

The pAD-2S-RBD-HA is a recombinant plasmid obtained by replacing the restriction enzyme sites of BsmBI of pAD3000 with 2S-RBD-HA gene segments shown in 12 th to 3373 th sites of SEQ ID NO.17 and keeping other sequences of pAD3000 unchanged.

2. pAD-PB2, pAD-PB1, pAD-PA, pAD-NP, pAD-M, pAD-NS, and pAD-NA were constructed as in example 5.

Second, rescue of recombinant SARS-CoV-2

The rescue method is the same as the second step of example 5, and is characterized in that pAD-S-RBD-HA is replaced by pAD-2S-RBD-HA, and the chicken embryo average HA titer range of the recombinant SARS-CoV-2 vaccine strain is 1:2⁷-1:2⁹。

Successfully obtains the recombinant SARS-CoV-2 vaccine strain of the chimeric pAD-2S-RBD-HA epitope, which is named as rFLU-2S-RBD-HA.

Identification of recombinant SARS-CoV-2

And identifying the rFLU-2S-RBD-HA, observing the virus morphology by an electron microscope, wherein most virus particles are spherical, few virus particles are filamentous, and the sizes of the virus particles are not completely consistent, wherein the spherical diameter is 80-120 nm, and the virus particles have envelope and accord with the typical morphology characteristics of influenza viruses. Inoculating the rFLU-2S-RBD-HA into SPF chick embryos of 9-11 days old, taking allantoic fluid of the second generation chick embryos to extract virus RNA, amplifying 8 gene segments (the amplification primers of each gene segment are shown in table 1 in the example) through RT-PCR, and sequencing, wherein the result is completely consistent with the expected gene sequence. The rFLU-2S-RBD-HA is subjected to mass culture of chick embryos, ultrafiltration concentration, sucrose gradient centrifugation purification, SDS-PAGE electrophoresis, gel staining and decoloration, and two clear bands with the sizes of about 60KD and 30KD can be observed, so that the existence of main components of the antigen is proved.

And fourthly, detecting the rFLU-S-RBD-HA, wherein the rFLU-S-RBD-HA HAs Temperature sensitive (ts), Cold adapted (ca) and Attenuated (att) phenotypes, and the detection method is the same as that of the example 5.

Fifth, in vivo immunization experiment in mice

The experimental immunization method was the same as that of example 1, except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-2S-RBD-HA", and as a result, as shown in Table 15, the antibody titers against SARS-CoV-2S protein and influenza virus detected in the serum after the secondary immunization were 1: 3560 and 1: 5120 it is shown that the vaccine can induce body to generate dual immune response against SARS-CoV-2 and influenza virus after immunization.

TABLE 15 rFLU-2S-RBD-HA Immunity Effect assay

Sixthly, attack toxicity test

The immunization and challenge protocol was the same as step six of example 1 except that "rFLU-SARS-CoV-2-NS" was replaced with "rFLU-2S-RBD-HA". The results show that: the PBS control group begins to develop symptoms of coronary pneumonia such as listlessness, hair erection, limited activity, small food intake, weight loss and the like 24h after being inoculated with culture solution containing SARS-CoV-2, and the virus titer in lung tissues reaches 10 in the third day after virus challenge^3.4TCID₅₀Per mL; on the third day after the mice in the rFLU-2S-RBD-HA group are attacked, the virus titer in lung tissues is not detected and is obviously reduced compared with that in a PBS control group. The rFLU-2S-RBD-HA HAs certain prevention effect on the novel coronavirus pneumonia.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> fifth medical center of general Hospital of the people's liberation force of China, Beijing Madlirabang Biotech Co., Ltd

<120> recombinant novel coronavirus, and preparation method and application thereof

<130> GNCFY200686

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 253

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

1 5 10 15

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

20 25 30

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

35 40 45

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

50 55 60

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

65 70 75 80

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

85 90 95

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

100 105 110

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

115 120 125

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

130 135 140

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

145 150 155 160

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

165 170 175

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

180 185 190

Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys

195 200 205

Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu

210 215 220

Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala

225 230 235 240

Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu

245 250

<210> 2

<211> 759

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aatattacaa acttgtgccc ttttggtgaa gtttttaacg ccaccagatt tgcatctgtt 60

tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt cctatataat 120

tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt aaatgatctc 180

tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt cagacaaatc 240

gctccagggc aaactggaaa gattgctgat tataattata aattaccaga tgattttaca 300

ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg taattataat 360

tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga tatttcaact 420

gaaatctatc aggccggtag cacaccttgt aatggtgttg aaggttttaa ttgttacttt 480

cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc atacagagta 540

gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc taaaaagtct 600

actaatttgg ttaaaaacaa atgtgtcaat ttcaacttca atggtttaac aggcacaggt 660

gttcttactg agtctaacaa aaagtttctg cctttccaac aatttggcag agacattgct 720

gacactactg atgctgtccg tgatccacag acacttgag 759

<210> 3

<211> 564

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

1 5 10 15

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

20 25 30

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

35 40 45

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

50 55 60

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

65 70 75 80

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

85 90 95

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

100 105 110

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

115 120 125

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

130 135 140

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

145 150 155 160

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

165 170 175

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

180 185 190

Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys

195 200 205

Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu

210 215 220

Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala

225 230 235 240

Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Gly Ser Gly

245 250 255

Gly Ser Gly Ser Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu

260 265 270

Ile Val Asn Ser Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu

275 280 285

Val Thr Leu Ala Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys

290 295 300

Asn Ile Val Asn Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser

305 310 315 320

Arg Val Lys Asn Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val Gly

325 330 335

Ser Gly Gly Ser Gly Ser Met Ala Asp Ser Asn Gly Thr Ile Thr Val

340 345 350

Glu Glu Leu Lys Lys Leu Leu Glu Gln Trp Asn Leu Val Ile Gly Phe

355 360 365

Leu Phe Leu Thr Trp Ile Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg

370 375 380

Asn Arg Phe Leu Tyr Ile Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp

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Pro Val Thr Leu Ala Cys Phe Val Leu Ala Ala Val Tyr Arg Ile Asn

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Trp Ile Thr Gly Gly Ile Ala Ile Ala Met Ala Cys Leu Val Gly Leu

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Met Trp Leu Ser Tyr Phe Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr

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Arg Ser Met Trp Ser Phe Asn Pro Glu Thr Asn Ile Leu Leu Asn Val

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Pro Leu His Gly Thr Ile Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu

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Val Ile Gly Ala Val Ile Leu Arg Gly His Leu Arg Ile Ala Gly His

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His Leu Gly Arg Cys Asp Ile Lys Asp Leu Pro Lys Glu Ile Thr Val

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Ala Thr Ser Arg Thr Leu Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg

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Val Ala Gly Asp Ser Gly Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly

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Asn Tyr Lys Leu Asn Thr Asp His Ser Ser Ser Ser Asp Asn Ile Ala

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Leu Leu Val Gln

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aatattacaa acttgtgccc ttttggtgaa gtttttaacg ccaccagatt tgcatctgtt 60

tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt cctatataat 120

tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt aaatgatctc 180

tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt cagacaaatc 240

gctccagggc aaactggaaa gattgctgat tataattata aattaccaga tgattttaca 300

ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg taattataat 360

tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga tatttcaact 420

gaaatctatc aggccggtag cacaccttgt aatggtgttg aaggttttaa ttgttacttt 480

cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc atacagagta 540

gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc taaaaagtct 600

actaatttgg ttaaaaacaa atgtgtcaat ttcaacttca atggtttaac aggcacaggt 660

gttcttactg agtctaacaa aaagtttctg cctttccaac aatttggcag agacattgct 720

gacactactg atgctgtccg tgatccacag acacttgagg gttcaggcgg atcggggagt 780

atgtactcat tcgtttcgga agagacaggt acgttaatag ttaatagcgt acttcttttt 840

cttgctttcg tggtattctt gctagttaca ctagccatcc ttactgcgct tcgattgtgt 900

gcgtactgct gcaatattgt taacgtgagt cttgtaaaac cttcttttta cgtttactct 960

cgtgttaaaa atctgaattc ttctagagtt cctgatcttc tggtcggttc aggcggatcg 1020

gggagtatgg cagattccaa cggtactatt accgttgaag agcttaaaaa gctccttgaa 1080

caatggaacc tagtaatagg tttcctattc cttacatgga tttgtcttct acaatttgcc 1140

tatgccaaca ggaataggtt tttgtatata attaagttaa ttttcctctg gctgttatgg 1200

ccagtaactt tagcttgttt tgtgcttgct gctgtttaca gaataaattg gatcaccggt 1260

ggaattgcta tcgcaatggc ttgtcttgta ggcttgatgt ggctcagcta cttcattgct 1320

tctttcagac tgtttgcgcg tacgcgttcc atgtggtcat tcaatccaga aactaacatt 1380

cttctcaacg tgccactcca tggcactatt ctgaccagac cgcttctaga aagtgaactc 1440

gtaatcggag ctgtgatcct tcgtggacat cttcgtattg ctggacacca tctaggacgc 1500

tgtgacatca aggacctgcc taaagaaatc actgttgcta catcacgaac gctttcttat 1560

tacaaattgg gagcttcgca gcgtgtagca ggtgactcag gttttgctgc atacagtcgc 1620

tacaggattg gcaactataa attaaacaca gaccattcca gtagcagtga caatattgct 1680

ttgcttgtac agtaa 1695

<210> 5

<211> 2620

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tattcgtctc agggagcaaa agcagggtga caaagacata atggatccta acactgtgtc 60

aagctttcag gtagattgct tcctttggca tgtccgcaaa caagttgcag accaagaact 120

aggtgatgcc ccattccttg atcggcttcg ccgagatcag aagtccctaa ggggaagagg 180

cagtactctc ggtctgaaca tcgaaacagc cacccgtgtt ggaaagcaga tagtggagag 240

gattctgaag gaagaatccg atgaggcact taaaatgacc atggcctccg cacctgcttc 300

gcgataccta actgacatga ctattgagga aatgtcaagg gactggttca tgctaatgcc 360

caagcagaaa gtggcaggcc ctctttgtat cagaatggac caggcaatca tggattaatg 420

aatattacaa acttgtgccc ttttggtgaa gtttttaacg ccaccagatt tgcatctgtt 480

tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt cctatataat 540

tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt aaatgatctc 600

tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt cagacaaatc 660

gctccagggc aaactggaaa gattgctgat tataattata aattaccaga tgattttaca 720

ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg taattataat 780

tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga tatttcaact 840

gaaatctatc aggccggtag cacaccttgt aatggtgttg aaggttttaa ttgttacttt 900

cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc atacagagta 960

gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc taaaaagtct 1020

actaatttgg ttaaaaacaa atgtgtcaat ttcaacttca atggtttaac aggcacaggt 1080

gttcttactg agtctaacaa aaagtttctg cctttccaac aatttggcag agacattgct 1140

gacactactg atgctgtccg tgatccacag acacttgagg gttcaggcgg atcggggagt 1200

atgtactcat tcgtttcgga agagacaggt acgttaatag ttaatagcgt acttcttttt 1260

cttgctttcg tggtattctt gctagttaca ctagccatcc ttactgcgct tcgattgtgt 1320

gcgtactgct gcaatattgt taacgtgagt cttgtaaaac cttcttttta cgtttactct 1380

cgtgttaaaa atctgaattc ttctagagtt cctgatcttc tggtctaagg ttcaggcgga 1440

tcggggagta tggcagattc caacggtact attaccgttg aagagcttaa aaagctcctt 1500

gaacaatgga acctagtaat aggtttccta ttccttacat ggatttgtct tctacaattt 1560

gcctatgcca acaggaatag gtttttgtat ataattaagt taattttcct ctggctgtta 1620

tggccagtaa ctttagcttg ttttgtgctt gctgctgttt acagaataaa ttggatcacc 1680

ggtggaattg ctatcgcaat ggcttgtctt gtaggcttga tgtggctcag ctacttcatt 1740

gcttctttca gactgtttgc gcgtacgcgt tccatgtggt cattcaatcc agaaactaac 1800

attcttctca acgtgccact ccatggcact attctgacca gaccgcttct agaaagtgaa 1860

ctcgtaatcg gagctgtgat ccttcgtgga catcttcgta ttgctggaca ccatctagga 1920

cgctgtgaca tcaaggacct gcctaaagaa atcactgttg ctacatcacg aacgctttct 1980

tattacaaat tgggagcttc gcagcgtgta gcaggtgact caggttttgc tgcatacagt 2040

cgctacagga ttggcaacta taaattaaac acagaccatt ccagtagcag tgacaatatt 2100

gctttgcttg tacagtaaaa gaacatcata ttgaaagcga atttcagtgt gatttttgac 2160

cggctagaga ccctaatatt actaagggct ttcaccgaaa cgggagcaat tgttggcgaa 2220

atttcaccat tgccttctct tccaggacat actaatgagg atgtcaaaaa tgcaattggg 2280

gtcctcatcg gaggacttga atggaatgat aacacagttc gagtctctaa aactctacag 2340

agattcgctt ggagaagcag tgatgagaat gggagacctc cactcactcc aaaatagaaa 2400

cggaaaatgg cgagaacaat taggtcaaaa gttcgaagaa ataagatggc tgattgaaga 2460

agtgagacac aaattgaaga taacagagaa tagttttgag caaataacat ttatgcaagc 2520

cttacagcta ctatttgaag tggaacaaga gataagaact ttctcgtttc agcttattta 2580

atgataaaaa acacccttgt ttctactaat acgagacgat 2620

<210> 6

<211> 2226

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tattcgtctc agggagcgaa agcaggggtt taaaatgaat ccaaaccaaa agataataac 60

cattggttcg atctgtatga caattggaat ggctaactta atattacaaa ttggaaacat 120

aatctcaata tgggttagcc actcaattca aattggaaat caaagccaga ttgaaacatg 180

caatcaaagc gtcattactt atgaaaacaa cacttgggta aatcagatga atattacaaa 240

cttgtgccct tttggtgaag tttttaacgc caccagattt gcatctgttt atgcttggaa 300

caggaagaga atcagcaact gtgttgctga ttattctgtc ctatataatt ccgcatcatt 360

ttccactttt aagtgttatg gagtgtctcc tactaaatta aatgatctct gctttactaa 420

tgtctatgca gattcatttg taattagagg tgatgaagtc agacaaatcg ctccagggca 480

aactggaaag attgctgatt ataattataa attaccagat gattttacag gctgcgttat 540

agcttggaat tctaacaatc ttgattctaa ggttggtggt aattataatt acctgtatag 600

attgtttagg aagtctaatc tcaaaccttt tgagagagat atttcaactg aaatctatca 660

ggccggtagc acaccttgta atggtgttga aggttttaat tgttactttc ctttacaatc 720

atatggtttc caacccacta atggtgttgg ttaccaacca tacagagtag tagtactttc 780

ttttgaactt ctacatgcac cagcaactgt ttgtggacct aaaaagtcta ctaatttggt 840

taaaaacaaa tgtgtcaatt tcaacttcaa tggtttaaca ggcacaggtg ttcttactga 900

gtctaacaaa aagtttctgc ctttccaaca atttggcaga gacattgctg acactactga 960

tgctgtccgt gatccacaga cacttgagac caactttgct gctggacagt cagtggtttc 1020

cgtgaaatta gcgggcaatt cctctctctg ccctgttagt ggatgggcta tatacagtaa 1080

agacaacagt gtaagaatcg gttccaaggg ggatgtgttt gtcataaggg aaccattcat 1140

atcatgctct cccttggaat gcagaacctt cttcttgact caaggggcct tgctaaatga 1200

caaacattcc aatggaacca ttaaagacag gagcccatac cgaaccctaa tgagctgtcc 1260

tattggtgaa gttccctctc catacaactc aagatttgag tcagtcgctt ggtcagcaag 1320

tgcttgtcat gatggcatca attggctaac aattggaatt tctggcccag acagtggggc 1380

agtggctgtg ttaaagtaca atggcataat aacagacact atcaagagtt ggaggaacaa 1440

tatattgaga acacaagagt ctgaatgtgc atgtgtaaat ggttcttgct ttaccataat 1500

gaccgatgga ccaagtgatg gacaggcctc atacaaaatc ttcagaatag aaaagggaaa 1560

gataatcaaa tcagtcgaaa tgaaagcccc taattatcac tatgaggaat gctcctgtta 1620

ccctgattct agtgaaatca catgtgtgtg cagggataac tggcatggct cgaatcgacc 1680

gtgggtgtct ttcaaccaga atctggaata tcagatggga tacatatgca gtggggtttt 1740

cggagacaat ccacgcccta atgataagac aggcagttgt ggtccagtat cgtctaatgg 1800

agcaaatgga gtaaaaggat tttcattcaa atacggcaat ggtgtttgga tagggagaac 1860

taaaagcatt agttcaagaa aaggttttga gatgatttgg gatccgaatg gatggactgg 1920

gactgacaat aaattctcaa taaagcaaga tatcgtagga ataaatgagt ggtcagggta 1980

tagcgggagt tttgttcagc atccagaact aacagggctg gattgtataa gaccttgctt 2040

ctgggttgaa ctaataagag ggcgacccga agagaacaca atctggacta gcgggagcag 2100

catatccttt tgtggtgtaa acagtgacac tgtgggttgg tcttggccag acggtgctga 2160

gttgccattt accattgaca agtaatctgt tcaaaaaact ccttgtttct actaatacga 2220

gacgat 2226

<210> 7

<211> 2606

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tattcgtctc agggagcaaa agcaggggaa aataaaaaca accaaaatga aggcaatact 60

agtagttctg ctatatacat ttacaaccgc aaatgcagca gcagcacctg gagcagcaaa 120

tattacaaac ttgtgccctt ttggtgaagt ttttaacgcc accagatttg catctgttta 180

tgcttggaac aggaagagaa tcagcaactg tgttgctgat tattctgtcc tatataattc 240

cgcatcattt tccactttta agtgttatgg agtgtctcct actaaattaa atgatctctg 300

ctttactaat gtctatgcag attcatttgt aattagaggt gatgaagtca gacaaatcgc 360

tccagggcaa actggaaaga ttgctgatta taattataaa ttaccagatg attttacagg 420

ctgcgttata gcttggaatt ctaacaatct tgattctaag gttggtggta attataatta 480

cctgtataga ttgtttagga agtctaatct caaacctttt gagagagata tttcaactga 540

aatctatcag gccggtagca caccttgtaa tggtgttgaa ggttttaatt gttactttcc 600

tttacaatca tatggtttcc aacccactaa tggtgttggt taccaaccat acagagtagt 660

agtactttct tttgaacttc tacatgcacc agcaactgtt tgtggaccta aaaagtctac 720

taatttggtt aaaaacaaat gtgtcaattt caacttcaat ggtttaacag gcacaggtgt 780

tcttactgag tctaacaaaa agtttctgcc tttccaacaa tttggcagag acattgctga 840

cactactgat gctgtccgtg atccacagac acttgaggca gcagcacctg gagcagcaga 900

cacattatgt ataggttatc atgcgaacaa ttcaacagac actgtagaca cagtactaga 960

aaagaatgta acagtaacac actctgttaa ccttctggaa gacaagcata acggaaaact 1020

atgcaaacta agaggggtag ccccattgca tttgggtaaa tgtaacattg ctggctggat 1080

cctgggaaat ccagagtgtg aatcactctc cacagcaagt tcatggtcct acattgtgga 1140

aacatctaat tcagacaatg gaacgtgtta cccaggagat ttcatcaatt atgaggagct 1200

aagagagcaa ttgagctcag tgtcatcatt tgaaaggttt gagatattcc ccaagacaag 1260

ttcatggccc aatcatgact cgaacaaagg tgtaacggca gcatgtcctc acgctggagc 1320

aaaaagcttc tacaaaaact tgatatggct agttaaaaaa ggaaattcat acccaaagct 1380

taaccaatcc tacattaatg ataaagggaa agaagtcctc gtgctgtggg gcattcacca 1440

tccatctact actgctgacc aacaaagtct ctatcagaat gcagatgcat atgtttttgt 1500

ggggacatca agatacagca agatgttcaa gccggaaata gcaacaagac ccaaagtgag 1560

ggatcgagaa gggagaatga actattactg gacactagta gagccgggag acaaaataac 1620

attcgaagca actggaaatc tagtggtacc gagatatgca ttcacaatgg aaagaaatgc 1680

tggatctggt attatcattt cagatacacc agtccacgat tgcaatacaa cttgtcagac 1740

acccgagggt gctataaaca ccagcctccc atttcagaat atacatccga tcacaattgg 1800

aaaatgtcca aagtatgtaa aaagcacaaa attgagactg gccacaggat tgaggaatgt 1860

tccgtctatt caatctagag gcctattcgg ggccattgcc ggcttcattg aaggggggtg 1920

gacagggatg gtagatggat ggtacggtta tcaccatcaa aatgagcagg ggtcaggata 1980

tgcagccgac ctgaagagca cacaaaatgc cattgacaag attactaaca aagtaaattc 2040

tgttattgaa aagatgaata cacagttcac agcagtgggt aaagagttca accacctgga 2100

aaaaagaata gagaatctaa ataaaaaagt tgatgatggt ttcctggaca tttggactta 2160

caatgccgaa ctgttggttc tattggaaaa tgaaagaact ttggactatc acgattcaaa 2220

tgtgaagaac ttgtatgaaa aagtaagaaa ccagttaaaa aacaatgcca aggaaattgg 2280

aaacggctgc tttgaatttt accacaaatg cgataacacg tgcatggaaa gtgtcaaaaa 2340

tggaacttat gactacccaa aatactcaga ggaagcaaaa ttaaacagag aaaaaataga 2400

tggggtaaag ctggaatcaa caaggattta ccagattttg gcgatctatt caactgtcgc 2460

cagttcattg gtactggtag tctccctggg ggcaatcagc ttctggatgt gctctaatgg 2520

gtctctacag tgtagaatat gtatttaaga ttagaatttc agagatatga ggaaaaacac 2580

ccttgtttct actaatacga gacgat 2606

<210> 8

<211> 2621

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tattcgtctc agggaagcaa aagcagggtg acaaaaacat aatggatcca aacactgtgt 60

caagctttca ggtagattgc tttctttggc atgtccgcaa acgagttgca gaccaagaac 120

taggtgatgc cccattcctt gatcggcttc gccgagatca gaaatcccta agaggaaggg 180

gcagcaccct cggtctggac atcgagacag ccacacgtgc tggaaagcag atagtggagc 240

ggattctgaa agaagaatcc gatgaggcac ttaaaatgac catggcctct gtacctgcgt 300

cgcgttacct aactgacatg actcttgagg aaatgtcaag ggactggtcc atgctcatac 360

ccaagcagaa agtggcaggc cctctttgta tcagaatgga ccaggcgatc atggattaat 420

gaatattaca aacttgtgcc cttttggtga agtttttaac gccaccagat ttgcatctgt 480

ttatgcttgg aacaggaaga gaatcagcaa ctgtgttgct gattattctg tcctatataa 540

ttccgcatca ttttccactt ttaagtgtta tggagtgtct cctactaaat taaatgatct 600

ctgctttact aatgtctatg cagattcatt tgtaattaga ggtgatgaag tcagacaaat 660

cgctccaggg caaactggaa agattgctga ttataattat aaattaccag atgattttac 720

aggctgcgtt atagcttgga attctaacaa tcttgattct aaggttggtg gtaattataa 780

ttacctgtat agattgttta ggaagtctaa tctcaaacct tttgagagag atatttcaac 840

tgaaatctat caggccggta gcacaccttg taatggtgtt gaaggtttta attgttactt 900

tcctttacaa tcatatggtt tccaacccac taatggtgtt ggttaccaac catacagagt 960

agtagtactt tcttttgaac ttctacatgc accagcaact gtttgtggac ctaaaaagtc 1020

tactaatttg gttaaaaaca aatgtgtcaa tttcaacttc aatggtttaa caggcacagg 1080

tgttcttact gagtctaaca aaaagtttct gcctttccaa caatttggca gagacattgc 1140

tgacactact gatgctgtcc gtgatccaca gacacttgag ggttcaggcg gatcggggag 1200

tatgtactca ttcgtttcgg aagagacagg tacgttaata gttaatagcg tacttctttt 1260

tcttgctttc gtggtattct tgctagttac actagccatc cttactgcgc ttcgattgtg 1320

tgcgtactgc tgcaatattg ttaacgtgag tcttgtaaaa ccttcttttt acgtttactc 1380

tcgtgttaaa aatctgaatt cttctagagt tcctgatctt ctggtctaag gttcaggcgg 1440

atcggggagt atggcagatt ccaacggtac tattaccgtt gaagagctta aaaagctcct 1500

tgaacaatgg aacctagtaa taggtttcct attccttaca tggatttgtc ttctacaatt 1560

tgcctatgcc aacaggaata ggtttttgta tataattaag ttaattttcc tctggctgtt 1620

atggccagta actttagctt gttttgtgct tgctgctgtt tacagaataa attggatcac 1680

cggtggaatt gctatcgcaa tggcttgtct tgtaggcttg atgtggctca gctacttcat 1740

tgcttctttc agactgtttg cgcgtacgcg ttccatgtgg tcattcaatc cagaaactaa 1800

cattcttctc aacgtgccac tccatggcac tattctgacc agaccgcttc tagaaagtga 1860

actcgtaatc ggagctgtga tccttcgtgg acatcttcgt attgctggac accatctagg 1920

acgctgtgac atcaaggacc tgcctaaaga aatcactgtt gctacatcac gaacgctttc 1980

ttattacaaa ttgggagctt cgcagcgtgt agcaggtgac tcaggttttg ctgcatacag 2040

tcgctacagg attggcaact ataaattaaa cacagaccat tccagtagca gtgacaatat 2100

tgctttgctt gtacagtaaa agaacatcat actgaaagcg aacttcagtg tgaattttga 2160

ccggctggag actctaatat tgctaagggc tttcaccgaa gagggagcaa ttgttggcga 2220

aatttcacca ttgccttctc ttccaggaca tactgctgag gatgtcaaaa atgcagttgg 2280

agtcctcatc gggggacttg aatggaatga taacacagtt cgagtctctg aaactctaca 2340

gagattcgct tggagaagca gtaatgagaa tgggagacct ccactcactc caaaacagaa 2400

acgagaaatg gcgggaacaa ttaggtcaga agtttgaaga aataagatgg ttgattgaag 2460

aagtgagaca caaactgaag ataacagaga atagttttga gcaaataaca tttatgcaag 2520

ccttacatct attgcttgaa gtggagcaag agataagaac tttctcgttt cagcttattt 2580

agtaataaaa aacacccttg tttctactaa tacgagacga t 2621

<210> 9

<211> 2199

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tattcgtctc agggagcgaa agcaggggtt taaaatgaat ccaaatcaga aaataacaac 60

cattggatca atctgtctgg tagtcggact aattagccta atattgcaaa tagggaatat 120

aatctcaata tggattagcc attcaattca aactggaagt caaaaccata ctggaatatg 180

caaccaaaac atcattacct ataaaaatag cacctgggta aaggacaata ttacaaactt 240

gtgccctttt ggtgaagttt ttaacgccac cagatttgca tctgtttatg cttggaacag 300

gaagagaatc agcaactgtg ttgctgatta ttctgtccta tataattccg catcattttc 360

cacttttaag tgttatggag tgtctcctac taaattaaat gatctctgct ttactaatgt 420

ctatgcagat tcatttgtaa ttagaggtga tgaagtcaga caaatcgctc cagggcaaac 480

tggaaagatt gctgattata attataaatt accagatgat tttacaggct gcgttatagc 540

ttggaattct aacaatcttg attctaaggt tggtggtaat tataattacc tgtatagatt 600

gtttaggaag tctaatctca aaccttttga gagagatatt tcaactgaaa tctatcaggc 660

cggtagcaca ccttgtaatg gtgttgaagg ttttaattgt tactttcctt tacaatcata 720

tggtttccaa cccactaatg gtgttggtta ccaaccatac agagtagtag tactttcttt 780

tgaacttcta catgcaccag caactgtttg tggacctaaa aagtctacta atttggttaa 840

aaacaaatgt gtcaatttca acttcaatgg tttaacaggc acaggtgttc ttactgagtc 900

taacaaaaag tttctgcctt tccaacaatt tggcagagac attgctgaca ctactgatgc 960

tgtccgtgat ccacagacac ttgagacaac ttcagtgata ttaaccggca attcatctct 1020

ttgtcccatc cgtgggtggg ctatatacag caaagacaat agcataagaa ttggttccaa 1080

aggagacgtt tttgtcataa gagagccctt tatttcatgt tctcacttgg aatgcaggac 1140

cttttttctg acccaaggtg ccttactgaa tgacaagcat tcaaatggga ctgttaagga 1200

cagaagccct tatagggcct taatgagctg ccctgtcggt gaagctccgt ccccgtacaa 1260

ttcaagattt gaatcggttg cttggtcagc aagtgcatgt catgatggca tgggctggct 1320

aacaatcgga atttcaggtc cagataatgg agcagtggct gtattaaaat acaacggcat 1380

aataactgaa accataaaaa gttggaggaa gaaaatattg aggacacaag agtctgaatg 1440

tgcctgtgta aatggttcat gttttactat aatgactgat ggcccgagtg atgggctggc 1500

ctcgtacaaa attttcaaga tcgaaaaggg gaaggttact aaatcaatag agttgaatgc 1560

acctaattct cactatgagg aatgttcctg ttaccctgat accggcaaag tgatgtgtgt 1620

gtgcagagac aactggcatg gttcgaaccg gccatgggtg tctttcgatc aaaacctgga 1680

ttatcaaata ggatacatct gcagtggggt tttcggtgac aacccgcgtc ccgaagatgg 1740

aacaggcagc tgtggtccag tgtatgttga tggagcaaac ggagtaaagg gattttcata 1800

taggtatggt aatggtgttt ggataggaag gaccaaaagt cacagttcca gacatgggtt 1860

tgagatgatt tgggatccta atggatggac agagactgat agtaagttct ctgttaggca 1920

agatgttgtg gcaatgactg attggtcagg gtatagcgga agtttcgttc aacatcctga 1980

gctaacaggg ctagactgta tgaggccgtg cttctgggtt gaattaatca ggggacgacc 2040

taaagaaaaa acaatctgga ctagtgcgag cagcatttct ttttgtggcg tgaatagtga 2100

tactgtagat tggtcttggc cagacggtgc tgagttgcca ttcagcattg acaagtagtc 2160

tgttcaaaaa actccttgtt tctactaata agagacgat 2199

<210> 10

<211> 2603

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tattcgtctc agggagcaaa agcaggggaa aataaaaaca accaaaatga aggcaaacct 60

actggtcctg ttatgtgcac ttgcagctgc agatgcagca gcagcacctg gagcagcaaa 120

tattacaaac ttgtgccctt ttggtgaagt ttttaacgcc accagatttg catctgttta 180

tgcttggaac aggaagagaa tcagcaactg tgttgctgat tattctgtcc tatataattc 240

cgcatcattt tccactttta agtgttatgg agtgtctcct actaaattaa atgatctctg 300

ctttactaat gtctatgcag attcatttgt aattagaggt gatgaagtca gacaaatcgc 360

tccagggcaa actggaaaga ttgctgatta taattataaa ttaccagatg attttacagg 420

ctgcgttata gcttggaatt ctaacaatct tgattctaag gttggtggta attataatta 480

cctgtataga ttgtttagga agtctaatct caaacctttt gagagagata tttcaactga 540

aatctatcag gccggtagca caccttgtaa tggtgttgaa ggttttaatt gttactttcc 600

tttacaatca tatggtttcc aacccactaa tggtgttggt taccaaccat acagagtagt 660

agtactttct tttgaacttc tacatgcacc agcaactgtt tgtggaccta aaaagtctac 720

taatttggtt aaaaacaaat gtgtcaattt caacttcaat ggtttaacag gcacaggtgt 780

tcttactgag tctaacaaaa agtttctgcc tttccaacaa tttggcagag acattgctga 840

cactactgat gctgtccgtg atccacagac acttgaggca gcagcacctg gagcagcaga 900

cacaatatgt ataggctacc atgcgaacaa ttcaaccgac actgttgaca cagtactcga 960

gaagaatgtg acagtgacac actctgttaa cctgctcgaa gacagccaca acggaaaact 1020

atgtagatta aaaggaatag ccccactaca attggggaaa tgtaacatcg ccggatggct 1080

cttgggaaac ccagaatgcg acccactgct tccagtgaga tcatggtcct acattgtaga 1140

aacaccaaac tctgagaatg gaatatgtta tccaggagat ttcatcgact atgaggagct 1200

gagggagcaa ttgagctcag tgtcatcatt cgaaagattc gaaatatttc ccaaagaaag 1260

ctcatggccc aaccacaaca caaacggagt aacggcagca tgctcccatg aggggaaaag 1320

cagtttttac agaaatttgc tatggctgac ggagaaggag ggctcatacc caaagctgaa 1380

aaattcttat gtgaacaaaa aagggaaaga agtccttgta ctgtggggta ttcatcaccc 1440

gcctaacagt aaggaacaac agaatctcta tcagaatgaa aatgcttatg tctctgtagt 1500

gacttcaaat tataacagga gatttacccc ggaaatagca gaaagaccca aagtaagaga 1560

tcaagctggg aggatgaact attactggac cttgctaaaa cccggagaca caataatatt 1620

tgaggcaaat ggaaatctaa tagcaccaat gtatgctttc gcactgagta gaggctttgg 1680

gtccggcatc atcacctcaa acgcatcaat gcatgagtgt aacacgaagt gtcaaacacc 1740

cctgggagct ataaacagca gtctccctta ccagaatata cacccagtca caataggaga 1800

gtgcccaaaa tacgtcagga gtgccaaatt gaggatggtt acaggactaa ggaacaatcc 1860

gtccattcaa tccagaggtc tatttggagc cattgccggt tttattgaag ggggatggac 1920

tggaatgata gatggatggt atggttatca tcatcagaat gaacagggat caggctatgc 1980

agcggatcaa aaaagcacac aaaatgccat taacgggatt acaaacaagg tgaacactgt 2040

tatcgagaaa atgaacattc aattcacagc tgtgggtaaa gaattcaaca aattagaaaa 2100

aaggatggaa aatttaaata aaaaagttga tgatggattt ctggacattt ggacatataa 2160

tgcagaattg ttagttctac tggaaaatga aaggactctg gatttccatg actcaaatgt 2220

gaagaatctg tatgagaaag taaaaagcca attaaagaat aatgccaaag aaatcggaaa 2280

tggatgtttt gagttctacc acaagtgtga caatgaatgc atggaaagtg taagaaatgg 2340

gacttatgat tatcccaaat attcagaaga gtcaaagttg aacagggaaa aggtagatgg 2400

agtgaaattg gaatcaatgg ggatctatca gattctggcg atctactcaa ctgtcgccag 2460

ttcactggtg cttttggtct ccctgggggc aatcagtttc tggatgtgtt ctaatggatc 2520

tttgcagtgc agaatatgca tctgagatta gaatttcaga gatatgagga aaaacaccct 2580

tgtttctact aataagagac gat 2603

<210> 11

<211> 627

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser

1 5 10 15

Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His

20 25 30

Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro

35 40 45

Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser

50 55 60

Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr

65 70 75 80

Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val

85 90 95

Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His

100 105 110

Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser

115 120 125

Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp

130 135 140

Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe

145 150 155 160

Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile

165 170 175

Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu

180 185 190

Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu

195 200 205

Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp

210 215 220

Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr

225 230 235 240

Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp

245 250 255

Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe

260 265 270

Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro

275 280 285

Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe

290 295 300

Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn

305 310 315 320

Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn

325 330 335

Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys

340 345 350

Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile

355 360 365

Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile

370 375 380

Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile

385 390 395 400

Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn

405 410 415

Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg

420 425 430

Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly

435 440 445

Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln

450 455 460

Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser

465 470 475 480

Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser

485 490 495

Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu

500 505 510

Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe

515 520 525

Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp

530 535 540

Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly

545 550 555 560

Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val

565 570 575

Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala

580 585 590

Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val

595 600 605

Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn

610 615 620

Ser Tyr Glu

625

<210> 12

<211> 1881

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggtgtttatt accctgacaa agttttcaga tcctcagttt tacattcaac tcaggacttg 60

ttcttacctt tcttttccaa tgttacttgg ttccatgcta tacatgtctc tgggaccaat 120

ggtactaaga ggtttgataa ccctgtccta ccatttaatg atggtgttta ttttgcttcc 180

actgagaagt ctaacataat aagaggctgg atttttggta ctactttaga ttcgaagacc 240

cagtccctac ttattgttaa taacgctact aatgttgtta ttaaagtctg tgaatttcaa 300

ttttgtaatg atccattttt gggtgtttat taccacaaaa acaacaaaag ttggatggaa 360

agtgagttca gagtttattc tagtgcgaat aattgcactt ttgaatatgt ctctcagcct 420

tttcttatgg accttgaagg aaaacagggt aatttcaaaa atcttaggga atttgtgttt 480

aagaatattg atggttattt taaaatatat tctaagcaca cgcctattaa tttagtgcgt 540

gatctccctc agggtttttc ggctttagaa ccattggtag atttgccaat aggtattaac 600

atcactaggt ttcaaacttt acttgcttta catagaagtt atttgactcc tggtgattct 660

tcttcaggtt ggacagctgg tgctgcagct tattatgtgg gttatcttca acctaggact 720

tttctattaa aatataatga aaatggaacc attacagatg ctgtagactg tgcacttgac 780

cctctctcag aaacaaagtg tacgttgaaa tccttcactg tagaaaaagg aatctatcaa 840

acttctaact ttagagtcca accaacagaa tctattgtta gatttcctaa tattacaaac 900

ttgtgccctt ttggtgaagt ttttaacgcc accagatttg catctgttta tgcttggaac 960

aggaagagaa tcagcaactg tgttgctgat tattctgtcc tatataattc cgcatcattt 1020

tccactttta agtgttatgg agtgtctcct actaaattaa atgatctctg ctttactaat 1080

gtctatgcag attcatttgt aattagaggt gatgaagtca gacaaatcgc tccagggcaa 1140

actggaaaga ttgctgatta taattataaa ttaccagatg attttacagg ctgcgttata 1200

gcttggaatt ctaacaatct tgattctaag gttggtggta attataatta cctgtataga 1260

ttgtttagga agtctaatct caaacctttt gagagagata tttcaactga aatctatcag 1320

gccggtagca caccttgtaa tggtgttgaa ggttttaatt gttactttcc tttacaatca 1380

tatggtttcc aacccactaa tggtgttggt taccaaccat acagagtagt agtactttct 1440

tttgaacttc tacatgcacc agcaactgtt tgtggaccta aaaagtctac taatttggtt 1500

aaaaacaaat gtgtcaattt caacttcaat ggtttaacag gcacaggtgt tcttactgag 1560

tctaacaaaa agtttctgcc tttccaacaa tttggcagag acattgctga cactactgat 1620

gctgtccgtg atccacagac acttgagatt cttgacatta caccatgttc ttttggtggt 1680

gtcagtgtta taacaccagg aacaaatact tctaaccagg ttgctgttct ttatcaggat 1740

gttaactgca cagaagtccc tgttgctatt catgcagatc aacttactcc tacttggcgt 1800

gtttattcta caggttctaa tgtttttcaa acacgtgcag gctgtttaat aggggctgaa 1860

catgtcaaca actcatatga g 1881

<210> 13

<211> 3345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tattcgtctc agggagcgaa agcaggggtt taaaatgaat ccaaaccaaa agataataac 60

cattggttcg atctgtatga caattggaat ggctaactta atattacaaa ttggaaacat 120

aatctcaata tgggttagcc actcaattca aattggaaat caaagccaga ttgaaacatg 180

caatcaaagc gtcattactt atgaaaacaa cacttgggta aatcagggtg tttattaccc 240

tgacaaagtt ttcagatcct cagttttaca ttcaactcag gacttgttct tacctttctt 300

ttccaatgtt acttggttcc atgctataca tgtctctggg accaatggta ctaagaggtt 360

tgataaccct gtcctaccat ttaatgatgg tgtttatttt gcttccactg agaagtctaa 420

cataataaga ggctggattt ttggtactac tttagattcg aagacccagt ccctacttat 480

tgttaataac gctactaatg ttgttattaa agtctgtgaa tttcaatttt gtaatgatcc 540

atttttgggt gtttattacc acaaaaacaa caaaagttgg atggaaagtg agttcagagt 600

ttattctagt gcgaataatt gcacttttga atatgtctct cagccttttc ttatggacct 660

tgaaggaaaa cagggtaatt tcaaaaatct tagggaattt gtgtttaaga atattgatgg 720

ttattttaaa atatattcta agcacacgcc tattaattta gtgcgtgatc tccctcaggg 780

tttttcggct ttagaaccat tggtagattt gccaataggt attaacatca ctaggtttca 840

aactttactt gctttacata gaagttattt gactcctggt gattcttctt caggttggac 900

agctggtgct gcagcttatt atgtgggtta tcttcaacct aggacttttc tattaaaata 960

taatgaaaat ggaaccatta cagatgctgt agactgtgca cttgaccctc tctcagaaac 1020

aaagtgtacg ttgaaatcct tcactgtaga aaaaggaatc tatcaaactt ctaactttag 1080

agtccaacca acagaatcta ttgttagatt tcctaatatt acaaacttgt gcccttttgg 1140

tgaagttttt aacgccacca gatttgcatc tgtttatgct tggaacagga agagaatcag 1200

caactgtgtt gctgattatt ctgtcctata taattccgca tcattttcca cttttaagtg 1260

ttatggagtg tctcctacta aattaaatga tctctgcttt actaatgtct atgcagattc 1320

atttgtaatt agaggtgatg aagtcagaca aatcgctcca gggcaaactg gaaagattgc 1380

tgattataat tataaattac cagatgattt tacaggctgc gttatagctt ggaattctaa 1440

caatcttgat tctaaggttg gtggtaatta taattacctg tatagattgt ttaggaagtc 1500

taatctcaaa ccttttgaga gagatatttc aactgaaatc tatcaggccg gtagcacacc 1560

ttgtaatggt gttgaaggtt ttaattgtta ctttccttta caatcatatg gtttccaacc 1620

cactaatggt gttggttacc aaccatacag agtagtagta ctttcttttg aacttctaca 1680

tgcaccagca actgtttgtg gacctaaaaa gtctactaat ttggttaaaa acaaatgtgt 1740

caatttcaac ttcaatggtt taacaggcac aggtgttctt actgagtcta acaaaaagtt 1800

tctgcctttc caacaatttg gcagagacat tgctgacact actgatgctg tccgtgatcc 1860

acagacactt gagattcttg acattacacc atgttctttt ggtggtgtca gtgttataac 1920

accaggaaca aatacttcta accaggttgc tgttctttat caggatgtta actgcacaga 1980

agtccctgtt gctattcatg cagatcaact tactcctact tggcgtgttt attctacagg 2040

ttctaatgtt tttcaaacac gtgcaggctg tttaataggg gctgaacatg tcaacaactc 2100

atatgagacc aactttgctg ctggacagtc agtggtttcc gtgaaattag cgggcaattc 2160

ctctctctgc cctgttagtg gatgggctat atacagtaaa gacaacagtg taagaatcgg 2220

ttccaagggg gatgtgtttg tcataaggga accattcata tcatgctctc ccttggaatg 2280

cagaaccttc ttcttgactc aaggggcctt gctaaatgac aaacattcca atggaaccat 2340

taaagacagg agcccatacc gaaccctaat gagctgtcct attggtgaag ttccctctcc 2400

atacaactca agatttgagt cagtcgcttg gtcagcaagt gcttgtcatg atggcatcaa 2460

ttggctaaca attggaattt ctggcccaga cagtggggca gtggctgtgt taaagtacaa 2520

tggcataata acagacacta tcaagagttg gaggaacaat atattgagaa cacaagagtc 2580

tgaatgtgca tgtgtaaatg gttcttgctt taccataatg accgatggac caagtgatgg 2640

acaggcctca tacaaaatct tcagaataga aaagggaaag ataatcaaat cagtcgaaat 2700

gaaagcccct aattatcact atgaggaatg ctcctgttac cctgattcta gtgaaatcac 2760

atgtgtgtgc agggataact ggcatggctc gaatcgaccg tgggtgtctt tcaaccagaa 2820

tctggaatat cagatgggat acatatgcag tggggttttc ggagacaatc cacgccctaa 2880

tgataagaca ggcagttgtg gtccagtatc gtctaatgga gcaaatggag taaaaggatt 2940

ttcattcaaa tacggcaatg gtgtttggat agggagaact aaaagcatta gttcaagaaa 3000

aggttttgag atgatttggg atccgaatgg atggactggg actgacaata aattctcaat 3060

aaagcaagat atcgtaggaa taaatgagtg gtcagggtat agcgggagtt ttgttcagca 3120

tccagaacta acagggctgg attgtataag accttgcttc tgggttgaac taataagagg 3180

gcgacccgaa gagaacacaa tctggactag cgggagcagc atatcctttt gtggtgtaaa 3240

cagtgacact gtgggttggt cttggccaga cggtgctgag ttgccattta ccattgacaa 3300

gtaatctgtt caaaaaactc cttgtttcta ctaatacgag acgat 3345

<210> 14

<211> 3728

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tattcgtctc agggagcaaa agcaggggaa aataaaaaca accaaaatga aggcaatact 60

agtagttctg ctatatacat ttacaaccgc aaatgcagca gcagcacctg gagcagcagg 120

tgtttattac cctgacaaag ttttcagatc ctcagtttta cattcaactc aggacttgtt 180

cttacctttc ttttccaatg ttacttggtt ccatgctata catgtctctg ggaccaatgg 240

tactaagagg tttgataacc ctgtcctacc atttaatgat ggtgtttatt ttgcttccac 300

tgagaagtct aacataataa gaggctggat ttttggtact actttagatt cgaagaccca 360

gtccctactt attgttaata acgctactaa tgttgttatt aaagtctgtg aatttcaatt 420

ttgtaatgat ccatttttgg gtgtttatta ccacaaaaac aacaaaagtt ggatggaaag 480

tgagttcaga gtttattcta gtgcgaataa ttgcactttt gaatatgtct ctcagccttt 540

tcttatggac cttgaaggaa aacagggtaa tttcaaaaat cttagggaat ttgtgtttaa 600

gaatattgat ggttatttta aaatatattc taagcacacg cctattaatt tagtgcgtga 660

tctccctcag ggtttttcgg ctttagaacc attggtagat ttgccaatag gtattaacat 720

cactaggttt caaactttac ttgctttaca tagaagttat ttgactcctg gtgattcttc 780

ttcaggttgg acagctggtg ctgcagctta ttatgtgggt tatcttcaac ctaggacttt 840

tctattaaaa tataatgaaa atggaaccat tacagatgct gtagactgtg cacttgaccc 900

tctctcagaa acaaagtgta cgttgaaatc cttcactgta gaaaaaggaa tctatcaaac 960

ttctaacttt agagtccaac caacagaatc tattgttaga tttcctaata ttacaaactt 1020

gtgccctttt ggtgaagttt ttaacgccac cagatttgca tctgtttatg cttggaacag 1080

gaagagaatc agcaactgtg ttgctgatta ttctgtccta tataattccg catcattttc 1140

cacttttaag tgttatggag tgtctcctac taaattaaat gatctctgct ttactaatgt 1200

ctatgcagat tcatttgtaa ttagaggtga tgaagtcaga caaatcgctc cagggcaaac 1260

tggaaagatt gctgattata attataaatt accagatgat tttacaggct gcgttatagc 1320

ttggaattct aacaatcttg attctaaggt tggtggtaat tataattacc tgtatagatt 1380

gtttaggaag tctaatctca aaccttttga gagagatatt tcaactgaaa tctatcaggc 1440

cggtagcaca ccttgtaatg gtgttgaagg ttttaattgt tactttcctt tacaatcata 1500

tggtttccaa cccactaatg gtgttggtta ccaaccatac agagtagtag tactttcttt 1560

tgaacttcta catgcaccag caactgtttg tggacctaaa aagtctacta atttggttaa 1620

aaacaaatgt gtcaatttca acttcaatgg tttaacaggc acaggtgttc ttactgagtc 1680

taacaaaaag tttctgcctt tccaacaatt tggcagagac attgctgaca ctactgatgc 1740

tgtccgtgat ccacagacac ttgagattct tgacattaca ccatgttctt ttggtggtgt 1800

cagtgttata acaccaggaa caaatacttc taaccaggtt gctgttcttt atcaggatgt 1860

taactgcaca gaagtccctg ttgctattca tgcagatcaa cttactccta cttggcgtgt 1920

ttattctaca ggttctaatg tttttcaaac acgtgcaggc tgtttaatag gggctgaaca 1980

tgtcaacaac tcatatgagg cagcagcacc tggagcagca gacacattat gtataggtta 2040

tcatgcgaac aattcaacag acactgtaga cacagtacta gaaaagaatg taacagtaac 2100

acactctgtt aaccttctgg aagacaagca taacggaaaa ctatgcaaac taagaggggt 2160

agccccattg catttgggta aatgtaacat tgctggctgg atcctgggaa atccagagtg 2220

tgaatcactc tccacagcaa gttcatggtc ctacattgtg gaaacatcta attcagacaa 2280

tggaacgtgt tacccaggag atttcatcaa ttatgaggag ctaagagagc aattgagctc 2340

agtgtcatca tttgaaaggt ttgagatatt ccccaagaca agttcatggc ccaatcatga 2400

ctcgaacaaa ggtgtaacgg cagcatgtcc tcacgctgga gcaaaaagct tctacaaaaa 2460

cttgatatgg ctagttaaaa aaggaaattc atacccaaag cttaaccaat cctacattaa 2520

tgataaaggg aaagaagtcc tcgtgctgtg gggcattcac catccatcta ctactgctga 2580

ccaacaaagt ctctatcaga atgcagatgc atatgttttt gtggggacat caagatacag 2640

caagatgttc aagccggaaa tagcaacaag acccaaagtg agggatcgag aagggagaat 2700

gaactattac tggacactag tagagccggg agacaaaata acattcgaag caactggaaa 2760

tctagtggta ccgagatatg cattcacaat ggaaagaaat gctggatctg gtattatcat 2820

ttcagataca ccagtccacg attgcaatac aacttgtcag acacccgagg gtgctataaa 2880

caccagcctc ccatttcaga atatacatcc gatcacaatt ggaaaatgtc caaagtatgt 2940

aaaaagcaca aaattgagac tggccacagg attgaggaat gttccgtcta ttcaatctag 3000

aggcctattc ggggccattg ccggcttcat tgaagggggg tggacaggga tggtagatgg 3060

atggtacggt tatcaccatc aaaatgagca ggggtcagga tatgcagccg acctgaagag 3120

cacacaaaat gccattgaca agattactaa caaagtaaat tctgttattg aaaagatgaa 3180

tacacagttc acagcagtgg gtaaagagtt caaccacctg gaaaaaagaa tagagaatct 3240

aaataaaaaa gttgatgatg gtttcctgga catttggact tacaatgccg aactgttggt 3300

tctattggaa aatgaaagaa ctttggacta tcacgattca aatgtgaaga acttgtatga 3360

aaaagtaaga aaccagttaa aaaacaatgc caaggaaatt ggaaacggct gctttgaatt 3420

ttaccacaaa tgcgataaca cgtgcatgga aagtgtcaaa aatggaactt atgactaccc 3480

aaaatactca gaggaagcaa aattaaacag agaaaaaata gatggggtaa agctggaatc 3540

aacaaggatt taccagattt tggcgatcta ttcaactgtc gccagttcat tggtactggt 3600

agtctccctg ggggcaatca gcttctggat gtgctctaat gggtctctac agtgtagaat 3660

atgtatttaa gattagaatt tcagagatat gaggaaaaac acccttgttt ctactaatac 3720

gagacgat 3728

<210> 15

<211> 2464

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tattcgtctc agggagcaaa agcagggtga caaagacata atggatccta acactgtgtc 60

aagctttcag gtagattgct tcctttggca tgtccgcaaa caagttgcag accaagaact 120

aggtgatgcc ccattccttg atcggcttcg ccgagatcag aagtccctaa ggggaagagg 180

cagtactctc ggtctgaaca tcgaaacagc cacccgtgtt ggaaagcaga tagtggagag 240

gattctgaag gaagaatccg atgaggcact taaaatgacc atggcctccg cacctgcttc 300

gcgataccta actgacatga ctattgagga aatgtcaagg gactggttca tgctaatgcc 360

caagcagaaa gtggcaggcc ctctttgtat cagaatggac caggcaatca tggattaatg 420

aatattacaa acttgtgccc ttttggtgaa gtttttaacg ccaccagatt tgcatctgtt 480

tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt cctatataat 540

tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt aaatgatctc 600

tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt cagacaaatc 660

gctccagggc aaactggaaa gattgctgat tataattata aattaccaga tgattttaca 720

ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg taattataat 780

tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga tatttcaact 840

gaaatctatc aggccggtag cacaccttgt aatggtgttg aaggttttaa ttgttacttt 900

cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc atacagagta 960

gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc taaaaagtct 1020

actaatttgg ttaaaaacaa atgtgtcaat ttcaacttca atggtttaac aggcacaggt 1080

gttcttactg agtctaacaa aaagtttctg cctttccaac aatttggcag agacattgct 1140

gacactactg atgctgtccg tgatccacag acacttgagg gttcaggcgg atcggggagt 1200

aatattacaa acttgtgccc ttttggtgaa gtttttaacg ccaccagatt tgcatctgtt 1260

tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt cctatataat 1320

tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt aaatgatctc 1380

tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt cagacaaatc 1440

gctccagggc aaactggaaa gattgctgat tataattata aattaccaga tgattttaca 1500

ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg taattataat 1560

tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga tatttcaact 1620

gaaatctatc aggccggtag cacaccttgt aatggtgttg aaggttttaa ttgttacttt 1680

cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc atacagagta 1740

gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc taaaaagtct 1800

actaatttgg ttaaaaacaa atgtgtcaat ttcaacttca atggtttaac aggcacaggt 1860

gttcttactg agtctaacaa aaagtttctg cctttccaac aatttggcag agacattgct 1920

gacactactg atgctgtccg tgatccacag acacttgagt aaaagaacat catattgaaa 1980

gcgaatttca gtgtgatttt tgaccggcta gagaccctaa tattactaag ggctttcacc 2040

gaaacgggag caattgttgg cgaaatttca ccattgcctt ctcttccagg acatactaat 2100

gaggatgtca aaaatgcaat tggggtcctc atcggaggac ttgaatggaa tgataacaca 2160

gttcgagtct ctaaaactct acagagattc gcttggagaa gcagtgatga gaatgggaga 2220

cctccactca ctccaaaata gaaacggaaa atggcgagaa caattaggtc aaaagttcga 2280

agaaataaga tggctgattg aagaagtgag acacaaattg aagataacag agaatagttt 2340

tgagcaaata acatttatgc aagccttaca gctactattt gaagtggaac aagagataag 2400

aactttctcg tttcagctta tttaatgata aaaaacaccc ttgtttctac taatacgaga 2460

cgat 2464

<210> 16

<211> 3003

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tattcgtctc agggagcgaa agcaggggtt taaaatgaat ccaaaccaaa agataataac 60

cattggttcg atctgtatga caattggaat ggctaactta atattacaaa ttggaaacat 120

aatctcaata tgggttagcc actcaattca aattggaaat caaagccaga ttgaaacatg 180

caatcaaagc gtcattactt atgaaaacaa cacttgggta aatcagaata ttacaaactt 240

gtgccctttt ggtgaagttt ttaacgccac cagatttgca tctgtttatg cttggaacag 300

gaagagaatc agcaactgtg ttgctgatta ttctgtccta tataattccg catcattttc 360

cacttttaag tgttatggag tgtctcctac taaattaaat gatctctgct ttactaatgt 420

ctatgcagat tcatttgtaa ttagaggtga tgaagtcaga caaatcgctc cagggcaaac 480

tggaaagatt gctgattata attataaatt accagatgat tttacaggct gcgttatagc 540

ttggaattct aacaatcttg attctaaggt tggtggtaat tataattacc tgtatagatt 600

gtttaggaag tctaatctca aaccttttga gagagatatt tcaactgaaa tctatcaggc 660

cggtagcaca ccttgtaatg gtgttgaagg ttttaattgt tactttcctt tacaatcata 720

tggtttccaa cccactaatg gtgttggtta ccaaccatac agagtagtag tactttcttt 780

tgaacttcta catgcaccag caactgtttg tggacctaaa aagtctacta atttggttaa 840

aaacaaatgt gtcaatttca acttcaatgg tttaacaggc acaggtgttc ttactgagtc 900

taacaaaaag tttctgcctt tccaacaatt tggcagagac attgctgaca ctactgatgc 960

tgtccgtgat ccacagacac ttgagggttc aggcggatcg gggagtaata ttacaaactt 1020

gtgccctttt ggtgaagttt ttaacgccac cagatttgca tctgtttatg cttggaacag 1080

gaagagaatc agcaactgtg ttgctgatta ttctgtccta tataattccg catcattttc 1140

cacttttaag tgttatggag tgtctcctac taaattaaat gatctctgct ttactaatgt 1200

ctatgcagat tcatttgtaa ttagaggtga tgaagtcaga caaatcgctc cagggcaaac 1260

tggaaagatt gctgattata attataaatt accagatgat tttacaggct gcgttatagc 1320

ttggaattct aacaatcttg attctaaggt tggtggtaat tataattacc tgtatagatt 1380

gtttaggaag tctaatctca aaccttttga gagagatatt tcaactgaaa tctatcaggc 1440

cggtagcaca ccttgtaatg gtgttgaagg ttttaattgt tactttcctt tacaatcata 1500

tggtttccaa cccactaatg gtgttggtta ccaaccatac agagtagtag tactttcttt 1560

tgaacttcta catgcaccag caactgtttg tggacctaaa aagtctacta atttggttaa 1620

aaacaaatgt gtcaatttca acttcaatgg tttaacaggc acaggtgttc ttactgagtc 1680

taacaaaaag tttctgcctt tccaacaatt tggcagagac attgctgaca ctactgatgc 1740

tgtccgtgat ccacagacac ttgagaccaa ctttgctgct ggacagtcag tggtttccgt 1800

gaaattagcg ggcaattcct ctctctgccc tgttagtgga tgggctatat acagtaaaga 1860

caacagtgta agaatcggtt ccaaggggga tgtgtttgtc ataagggaac cattcatatc 1920

atgctctccc ttggaatgca gaaccttctt cttgactcaa ggggccttgc taaatgacaa 1980

acattccaat ggaaccatta aagacaggag cccataccga accctaatga gctgtcctat 2040

tggtgaagtt ccctctccat acaactcaag atttgagtca gtcgcttggt cagcaagtgc 2100

ttgtcatgat ggcatcaatt ggctaacaat tggaatttct ggcccagaca gtggggcagt 2160

ggctgtgtta aagtacaatg gcataataac agacactatc aagagttgga ggaacaatat 2220

attgagaaca caagagtctg aatgtgcatg tgtaaatggt tcttgcttta ccataatgac 2280

cgatggacca agtgatggac aggcctcata caaaatcttc agaatagaaa agggaaagat 2340

aatcaaatca gtcgaaatga aagcccctaa ttatcactat gaggaatgct cctgttaccc 2400

tgattctagt gaaatcacat gtgtgtgcag ggataactgg catggctcga atcgaccgtg 2460

ggtgtctttc aaccagaatc tggaatatca gatgggatac atatgcagtg gggttttcgg 2520

agacaatcca cgccctaatg ataagacagg cagttgtggt ccagtatcgt ctaatggagc 2580

aaatggagta aaaggatttt cattcaaata cggcaatggt gtttggatag ggagaactaa 2640

aagcattagt tcaagaaaag gttttgagat gatttgggat ccgaatggat ggactgggac 2700

tgacaataaa ttctcaataa agcaagatat cgtaggaata aatgagtggt cagggtatag 2760

cgggagtttt gttcagcatc cagaactaac agggctggat tgtataagac cttgcttctg 2820

ggttgaacta ataagagggc gacccgaaga gaacacaatc tggactagcg ggagcagcat 2880

atccttttgt ggtgtaaaca gtgacactgt gggttggtct tggccagacg gtgctgagtt 2940

gccatttacc attgacaagt aatctgttca aaaaactcct tgtttctact aatacgagac 3000

gat 3003

<210> 17

<211> 3386

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tattcgtctc agggagcaaa agcaggggaa aataaaaaca accaaaatga aggcaatact 60

agtagttctg ctatatacat ttacaaccgc aaatgcagca gcagcacctg gagcagcaaa 120

tattacaaac ttgtgccctt ttggtgaagt ttttaacgcc accagatttg catctgttta 180

tgcttggaac aggaagagaa tcagcaactg tgttgctgat tattctgtcc tatataattc 240

cgcatcattt tccactttta agtgttatgg agtgtctcct actaaattaa atgatctctg 300

ctttactaat gtctatgcag attcatttgt aattagaggt gatgaagtca gacaaatcgc 360

tccagggcaa actggaaaga ttgctgatta taattataaa ttaccagatg attttacagg 420

ctgcgttata gcttggaatt ctaacaatct tgattctaag gttggtggta attataatta 480

cctgtataga ttgtttagga agtctaatct caaacctttt gagagagata tttcaactga 540

aatctatcag gccggtagca caccttgtaa tggtgttgaa ggttttaatt gttactttcc 600

tttacaatca tatggtttcc aacccactaa tggtgttggt taccaaccat acagagtagt 660

agtactttct tttgaacttc tacatgcacc agcaactgtt tgtggaccta aaaagtctac 720

taatttggtt aaaaacaaat gtgtcaattt caacttcaat ggtttaacag gcacaggtgt 780

tcttactgag tctaacaaaa agtttctgcc tttccaacaa tttggcagag acattgctga 840

cactactgat gctgtccgtg atccacagac acttgagggt tcaggcggat cggggagtaa 900

tattacaaac ttgtgccctt ttggtgaagt ttttaacgcc accagatttg catctgttta 960

tgcttggaac aggaagagaa tcagcaactg tgttgctgat tattctgtcc tatataattc 1020

cgcatcattt tccactttta agtgttatgg agtgtctcct actaaattaa atgatctctg 1080

ctttactaat gtctatgcag attcatttgt aattagaggt gatgaagtca gacaaatcgc 1140

tccagggcaa actggaaaga ttgctgatta taattataaa ttaccagatg attttacagg 1200

ctgcgttata gcttggaatt ctaacaatct tgattctaag gttggtggta attataatta 1260

cctgtataga ttgtttagga agtctaatct caaacctttt gagagagata tttcaactga 1320

aatctatcag gccggtagca caccttgtaa tggtgttgaa ggttttaatt gttactttcc 1380

tttacaatca tatggtttcc aacccactaa tggtgttggt taccaaccat acagagtagt 1440

agtactttct tttgaacttc tacatgcacc agcaactgtt tgtggaccta aaaagtctac 1500

taatttggtt aaaaacaaat gtgtcaattt caacttcaat ggtttaacag gcacaggtgt 1560

tcttactgag tctaacaaaa agtttctgcc tttccaacaa tttggcagag acattgctga 1620

cactactgat gctgtccgtg atccacagac acttgaggca gcagcacctg gagcagcaga 1680

cacattatgt ataggttatc atgcgaacaa ttcaacagac actgtagaca cagtactaga 1740

aaagaatgta acagtaacac actctgttaa ccttctggaa gacaagcata acggaaaact 1800

atgcaaacta agaggggtag ccccattgca tttgggtaaa tgtaacattg ctggctggat 1860

cctgggaaat ccagagtgtg aatcactctc cacagcaagt tcatggtcct acattgtgga 1920

aacatctaat tcagacaatg gaacgtgtta cccaggagat ttcatcaatt atgaggagct 1980

aagagagcaa ttgagctcag tgtcatcatt tgaaaggttt gagatattcc ccaagacaag 2040

ttcatggccc aatcatgact cgaacaaagg tgtaacggca gcatgtcctc acgctggagc 2100

aaaaagcttc tacaaaaact tgatatggct agttaaaaaa ggaaattcat acccaaagct 2160

taaccaatcc tacattaatg ataaagggaa agaagtcctc gtgctgtggg gcattcacca 2220

tccatctact actgctgacc aacaaagtct ctatcagaat gcagatgcat atgtttttgt 2280

ggggacatca agatacagca agatgttcaa gccggaaata gcaacaagac ccaaagtgag 2340

ggatcgagaa gggagaatga actattactg gacactagta gagccgggag acaaaataac 2400

attcgaagca actggaaatc tagtggtacc gagatatgca ttcacaatgg aaagaaatgc 2460

tggatctggt attatcattt cagatacacc agtccacgat tgcaatacaa cttgtcagac 2520

acccgagggt gctataaaca ccagcctccc atttcagaat atacatccga tcacaattgg 2580

aaaatgtcca aagtatgtaa aaagcacaaa attgagactg gccacaggat tgaggaatgt 2640

tccgtctatt caatctagag gcctattcgg ggccattgcc ggcttcattg aaggggggtg 2700

gacagggatg gtagatggat ggtacggtta tcaccatcaa aatgagcagg ggtcaggata 2760

tgcagccgac ctgaagagca cacaaaatgc cattgacaag attactaaca aagtaaattc 2820

tgttattgaa aagatgaata cacagttcac agcagtgggt aaagagttca accacctgga 2880

aaaaagaata gagaatctaa ataaaaaagt tgatgatggt ttcctggaca tttggactta 2940

caatgccgaa ctgttggttc tattggaaaa tgaaagaact ttggactatc acgattcaaa 3000

tgtgaagaac ttgtatgaaa aagtaagaaa ccagttaaaa aacaatgcca aggaaattgg 3060

aaacggctgc tttgaatttt accacaaatg cgataacacg tgcatggaaa gtgtcaaaaa 3120

tggaacttat gactacccaa aatactcaga ggaagcaaaa ttaaacagag aaaaaataga 3180

tggggtaaag ctggaatcaa caaggattta ccagattttg gcgatctatt caactgtcgc 3240

cagttcattg gtactggtag tctccctggg ggcaatcagc ttctggatgt gctctaatgg 3300

gtctctacag tgtagaatat gtatttaaga ttagaatttc agagatatga ggaaaaacac 3360

ccttgtttct actaatacga gacgat 3386

Claims

1. A recombinant virus prepared according to a method comprising the steps of: co-transfecting host cells with a recombinant plasmid containing a PB2 gene of an influenza virus, a recombinant plasmid containing a PB1 gene of the influenza virus, a recombinant plasmid containing a PA gene of the influenza virus, a recombinant plasmid containing an NP gene of the influenza virus, a recombinant plasmid containing an M gene of the influenza virus, a recombinant plasmid containing an NS gene of the influenza virus, a recombinant plasmid containing an HA gene of the influenza virus and a recombinant plasmid containing an NA gene of the influenza virus, and culturing to obtain the recombinant virus; the method is characterized in that:

the NS gene of the influenza virus is replaced by a recombinant DNA molecule named as S-RBD + E + M-NS gene;

the S-RBD + E + M-NS gene is a recombinant DNA molecule obtained by inserting an S-RBD + E + M gene between the 375 nd to the 376 nd nucleotides from the 5 ' end of an open reading frame of the NS gene of the influenza virus, connecting the 375 nd nucleotides from the 5 ' end of the open reading frame of the NS gene of the influenza virus and the S-RBD + E + M gene through 5 ' -TAATG-3 ', connecting the S-RBD + E + M gene and the 376 nd nucleotides from the 5 ' end of the open reading frame of the NS gene of the influenza virus, and keeping other nucleotides of the NS gene of the influenza virus unchanged;

the S-RBD + E + M gene is a receptor binding region gene for coding SARS-CoV-2S protein and a DNA molecule formed by connecting an E protein gene and an M protein gene;

the nucleotide sequence of the S-RBD + E + M-NS gene is 12 th to 2604 th sites of SEQ ID NO.5 or 12 th to 2605 th sites of SEQ ID NO. 8;

the influenza virus PB2, the influenza virus PB1, the influenza virus PA, the influenza virus NP, the influenza virus M and the influenza virus NS are cold-adapted attenuated influenza virus strains A/AnnArbor/6/60; the influenza virus in the HA of the influenza virus and the NA of the influenza virus is influenza virus strain A/Michigan/45/2015;

or the influenza virus in PB2, PB1, PA, NP, M, NS, HA and NA of the influenza virus is influenza virus strain A/PR/8/1934.

2. Use of the recombinant virus of claim 1 in the preparation of a product for the prevention of diseases caused by influenza virus and/or SARS-CoV-2.

3. A chimeric vaccine produced from the recombinant virus of claim 1.

4. Use of the chimeric vaccine of claim 3 for the preparation of a product for the prevention of diseases caused by influenza virus and/or SARS-CoV-2.