CN111206022A

CN111206022A - Recombinant virus for expressing Lassa fever virus empty capsid and preparation method thereof

Info

Publication number: CN111206022A
Application number: CN202010103832.1A
Authority: CN
Inventors: 赵忠鹏; 李靖; 李敏; 李霄; 焦俊; 杨晓岚; 谷宏婧; 张真; 申梦; 刘媛
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS; Academy of Military Medical Sciences AMMS of PLA
Priority date: 2020-02-20
Filing date: 2020-02-20
Publication date: 2020-05-29

Abstract

The invention discloses a recombinant virus for expressing a Lassa fever virus empty capsid and a preparation method thereof. The invention firstly discloses a construction method of recombinant viruses, which comprises the following steps: co-transfecting the transfer plasmid and the poxvirus to a host cell to obtain a recombinant virus; the transfer plasmid contains recombinant DNA molecules constructed based on the upstream and downstream homology arms of the poxvirus-stringent nonessential region genes, including the structural protein Z, GPC of Lassa fever virus and the NP gene. The invention further discloses the recombinant virus obtained by the construction and application thereof. The recombinant virus improves the safety of clinical application by knocking out a strict nonessential region, can stably exist and efficiently generates the lassa fever virus empty capsid in the whole process of early and late stages; the vaccine is used for immunizing mice and rhesus monkeys, and can generate high-titer anti-Lassa fever virus antibodies to block infection of Lassa fever virus pseudoviruses on sensitive cells.

Description

Recombinant virus for expressing Lassa fever virus empty capsid and preparation method thereof

Technical Field

The present invention relates to the fields of immunology and genetic engineering. In particular to a recombinant virus for expressing the empty capsid of the Lassa fever virus and a preparation method thereof.

Background

Lassa feber (Lassa lever) is an acute virulent viral (LASV) infectious disease, and according to statistics, 20 to 50 million people infected per year cause 0.5 to 1 million people to die, and the threatened population reaches 3700 million. There is currently no commercial lassa fever vaccine approved, and numerous lassa fever vaccine candidates are still in the laboratory research phase.

LASV is a two-segmented, single-stranded negative-strand RNA with 3.4kb S-strand and 7.2kb L-strand, respectively, that encodes 5 structural proteins. Two genes of the S chain encode the nucleoprotein NP, the glycoproteins GP1 and GP 2. Two genes of the L chain encode the viral polymerase protein (L) and the Z protein. GP1 and GP2 proteins are produced by post-translational cleavage of the precursor glycoprotein GPC encoded by G gene and constitute surface fibers in tetrameric form, which are involved in virus neutralization. LASV is divided into five genotypes and a plurality of serotypes according to GPC and NP genes, and different types of viruses can have certain cross protection but cannot achieve complete protection.

The application of virus-like particles and recombinant viral vectors as vaccines is gradually commercialized, and particularly, the application of the virus-like particles and the recombinant viral vectors is particularly emphasized in acute virulent virus vaccines with high requirements on biological safety.

With the development of genetic engineering technology, especially in 1982, Paoletti and Moss successfully applied vaccinia virus as a vector to express foreign genes in mammals for the first time, the vector was widely used for the research of recombinant live virus vector vaccines. In 1987, the recombinant vaccinia virus expressing rabies virus G gene was first approved for use in wild animals and successfully controlled rabies in wild animals in europe and north america; in 1994, the mass production of recombinant vaccinia virus vaccines expressing the genes for HN and F proteins of Newcastle disease virus was approved in the United states as the first commercial live vector vaccine; the American Ministry of agriculture formally approved the commercialization of the recombinant fowlpox virus live vector vaccine VAX FP-N in 1995.

With the successful application of recombinant poxviruses in the veterinary medicine field, there is a trend to develop poxvirus vectors with host specificity, such as canarypox virus, suipoxvirus, capripoxvirus, etc.; in the field of medicine, poxvirus is one of the hot spots of recombinant virus vectors due to its important economic value and good safety, a phase I clinical test of recombinant vaccinia virus Aldesleukin for treating malignant melanoma is started in the United states in 1996, and a phase II clinical test of the project is completed in 2001, so far, hundreds of researches using recombinant vaccinia virus as a biological agent are in the stage of medical clinical tests in the world, and the clinical batches are obtained in T601 (recombinant oncolytic vaccinia virus injection) 4 months in 2019, relating to the fields of prevention and treatment of various diseases such as cancer, virulent infectious diseases and the like.

The vaccinia virus genome consists of linear, double-stranded DNA, with an AT content of about 75%, and no infectivity of viral nucleic acids. Like other poxviruses, the vaccinia virus genome comprises a central coding region and inverted terminal repeats identical at both ends, encoding 147 open reading frames at a density of 93%. The vaccinia virus has the advantages of being safe as a preventive or therapeutic biological agent proved by a large number of animal and human experiments; the genome capacity is large, and a 25Kbp exogenous gene can be inserted; the protein expressed by the recombinant poxvirus has the processing, modification and transfer processes, and the antigenicity is better preserved; the host range is wide, the poxvirus can infect most mammals including human, mouse, etc., and can also be propagated in corresponding vaccine production cell lines, and the poxvirus is particularly suitable to be used as a carrier for preventing zoonosis and natural epidemic infectious diseases. In addition, the poxvirus is particularly suitable for popularization and use in developing countries, is stable, has low freeze-drying cost and is easy to store, so that the poxvirus is easy to produce and use, and the recombinant vaccinia virus vaccine is easy to distinguish natural infection and immunization individuals.

At present, there is no candidate vaccine of the lassa fever virus empty capsid which takes recombinant virus as a vector, the vaccine can cross-protect the infection of the lassa fever virus of any genotype or serotype, and the vaccine for protecting the infection of the lassa fever virus of various genotypes or serotypes has not been reported.

Disclosure of Invention

The technical problem to be solved by the invention is to obtain the recombinant vaccinia virus expressing the lassa fever virus empty capsid, and particularly to protect the infection of the lassa fever virus with various genotypes or serotypes after the expressed lassa fever virus empty capsid is used for immunizing animals.

In order to solve the technical problems, the invention firstly provides a construction method of a recombinant virus.

The construction method of the recombinant virus comprises the following steps:

co-transfecting the transfer plasmid and the poxvirus to a host cell to obtain a recombinant virus;

the transfer plasmid contains recombinant DNA molecules which are constructed on the basis of upstream and downstream homology arms of the genes of the non-essential regions strict by the poxvirus, and the recombinant DNA molecules comprise a Lassa fever virus structural protein Z gene, a Lassa fever virus structural protein GPC gene and a Lassa fever virus structural protein NP gene.

In the construction method, the Lassa fever virus structural protein Z gene, the Lassa fever virus structural protein GPC gene and the Lassa fever virus structural protein NP gene are all positioned between the upstream and downstream homologous arms of the strict nonessential region gene of the poxvirus. Thus, the structural protein Z gene of the lassa fever virus, the structural protein GPC gene of the lassa fever virus and the structural protein NP gene of the lassa fever virus can be inserted between the upstream and downstream homology arms of the strict nonessential region gene of the poxvirus by homologous recombination. The structural protein Z gene of the lassa fever virus, the structural protein GPC gene of the lassa fever virus and the structural protein NP gene of the lassa fever virus are constructed in independent expression cassettes, the promoter of each expression cassette can be PE/L, and the terminator can be T5 nT.

In the construction method, the amino acid sequence of the protein coded by the structural protein Z gene of the lassa fever virus is SEQ ID NO.2, the amino acid sequence of the protein coded by the structural protein GPC gene of the lassa fever virus is SEQ ID NO.3, and the amino acid sequence of the protein coded by the structural protein NP gene of the lassa fever virus is SEQ ID NO. 4.

In the above construction method, the coding sequence of the structural protein Z gene of the Lassa fever virus is the reverse complementary sequence of the 508 nd-807 th position of SEQ ID NO.1, the coding sequence of the structural protein GPC gene of the Lassa fever virus is the 888 nd 2363 th position of SEQ ID NO.1 and the coding sequence of the structural protein NP gene of the Lassa fever virus is the 2412 nd-4088 nd position of SEQ ID NO. 1.

In the construction method, the sequence of each gene in the recombinant DNA molecule is Lassa fever virus structural protein Z gene, Lassa fever virus structural protein GPC gene and Lassa fever virus structural protein NP gene in sequence.

In the above construction method, the recombinant DNA molecule further comprises two loxp sites and a selectable marker protein gene located between the loxp sites. The screening marker protein gene is constructed in an independent expression cassette, the promoter of the expression cassette is PE/L, the terminator is T5nT, and the loxp sites are respectively positioned at two sides of the expression cassette.

Specifically, the screening marker protein gene may be a green fluorescent protein gene, a red fluorescent protein gene or a yellow fluorescent protein gene. In a specific embodiment of the invention, the sequence of the loxp site is shown in position 4096-4129 of SEQ ID NO.1, and the selection marker protein gene is the green fluorescent protein (EGFP) gene shown in position 4170-4889 of SEQ ID NO. 1.

In the above construction method, the construction method further comprises co-transfecting host cells with the pVAX1-Cre plasmid and the recombinant virus to obtain the recombinant virus without the selection marker protein gene.

According to the invention, the knock-out of the exogenous screening marker EGFP gene is realized by utilizing a Cre/Loxp system.

In the above construction method, the non-essential region gene of the poxvirus stringent is thymidine kinase TK gene. In a specific embodiment of the invention, the nucleotide sequence of the upstream homology arm of the thymidine kinase TK gene is shown as 1 st-500 th position in SEQ ID NO.1, and the nucleotide sequence of the downstream homology arm of the thymidine kinase TK gene is shown as 4930 nd-5945 th position.

In the above construction method, the poxvirus may be a vaccinia virus Tiantan strain.

In the construction method, the host cell is MRC-5(NO. CCL171) cell, Vero (NO. CCL81) cell, chick embryo primary epithelial cell or BHK-21(NO. CCL10) cell.

The recombinant virus constructed by the construction method is also within the protection scope of the invention.

The invention further provides recombinant lassa fever virus-like particles (i.e., lassa fever virus empty capsids) produced by recombinant viruses.

The application of the recombinant lassa fever virus-like particle in preparing lassa fever vaccines is also within the protection scope of the invention.

The application of the recombinant lassa fever virus-like particles in preparing the products for preventing and/or treating lassa fever is also within the protection scope of the invention.

The recombinant Lassa fever virus-like particle is applied to preparing products for preventing and/or treating diseases caused by poxvirus.

Wherein the poxvirus may be a virus of the sub-genus vaccinia virus, the virus of the sub-genus vaccinia virus includes variola virus, vaccinia virus and monkeypox virus, the disease caused by variola virus is variola, and the disease caused by monkeypox virus is monkeypox.

The invention further provides a transfer vector.

The transfer plasmid contains recombinant DNA molecules constructed on the basis of upstream and downstream homology arms of genes of a non-essential region strictly controlled by poxvirus, and the recombinant DNA molecules comprise a Lassa fever virus structural protein Z gene, a Lassa fever virus structural protein GPC gene and a Lassa fever virus structural protein NP gene.

In the transfer plasmid, the amino acid sequence of the protein coded by the structural protein Z gene of the lassa fever virus is shown as SEQ ID NO.2, the amino acid sequence of the protein coded by the structural protein GPC gene of the lassa fever virus is shown as SEQ ID NO.3, and the amino acid sequence of the protein coded by the structural protein NP gene of the lassa fever virus is shown as SEQ ID NO. 4.

In the above transfer plasmid, the coding sequence of the structural protein Z gene of the lassa fever virus is the reverse complement sequence of the 508 nd-807 th position of SEQ ID NO.1, the coding sequence of the structural protein GPC gene of the lassa fever virus is the 888 nd 2363 th position of SEQ ID NO.1 and the coding sequence of the structural protein NP gene of the lassa fever virus is the 2412 nd-4088 nd position of SEQ ID NO. 1.

In the transfer plasmid, the sequence of each gene in the recombinant DNA molecule is Lassa fever virus structural protein Z gene, Lassa fever virus structural protein GPC gene and Lassa fever virus structural protein NP gene in sequence.

In the above transfer plasmid, the recombinant DNA molecule further comprises two loxp sites and a selectable marker protein gene located between the loxp sites. Specifically, the screening marker protein gene may be a green fluorescent protein gene, a red fluorescent protein gene or a yellow fluorescent protein gene. In a specific embodiment of the invention, the sequence of the loxp site is shown as position 4096-4129 (which is the same as the sequence shown as position 4897-4930) in SEQ ID NO.1, and the selection marker protein gene is the green fluorescent protein gene (EGFP) shown as position 4170-4889 in SEQ ID NO. 1.

In a specific embodiment of the invention, the transfer plasmid is a transfer plasmid pBSTKLSVLP, the transfer plasmid takes pBluescript as a skeleton vector, is cloned into a recombinant DNA molecule constructed on the basis of the upstream and downstream homologous arms of a gene of a strict nonessential region of poxvirus, namely thymidine kinase TK gene, to construct a transfer plasmid, four independent exogenous gene expression cassettes which take PE/L as a promoter and T5nT as a terminator are sequentially inserted in the middle of the transfer plasmid, and the exogenous genes are respectively optimized genes of structural protein Z, GPC and NP of the lassa fever virus and genes of exogenous screening marker protein EGFP (wherein, both sides of the gene expression cassette of the screening marker protein EGFP also contain Loxp genes), and respectively express structural protein Z, GPC, NP and the screening marker protein EGFP.

The partial nucleotide sequence of the transfer plasmid pBSTKLSVLP is shown as SEQ ID NO.1, wherein, the 1 st to 500 th sites of SEQ ID NO.1 are an upstream homologous arm L sequence of TK gene, the 501 st and 507 th sites of SEQ ID NO.1 are reverse complementary sequences of a terminator T5nT sequence, the 508 th and 807 th sites of SEQ ID NO.1 are reverse complementary sequences of the optimized structural protein Z of the lassa fever virus, the 808 th and 847 th sites of SEQ ID NO.1 are reverse complementary sequences of a promoter PE/L sequence, the 1 st and 887 th sites of SEQ ID NO.1 are promoter PE/L sequence, the 1 st and 888 nd 2363 st sites of SEQ ID NO.1 are the optimized structural protein GPC of the lassa fever virus, the 1 st and 2364 nd 2370 th sites of SEQ ID NO.1 are terminator T5nT sequence, the 1 st and the 232410 th sites of SEQ ID NO.1 are promoter PE/L sequence, and the 1 st and 4088 th site of the optimized structural protein NP 1 st and 2412 nd sites of the optimized structural protein NP 1 of the SEQ ID NO.1 st and the optimized structural protein NP 2 nd sites of the SEQ ID NO.1 4088 is the coding sequence of the optimized structural protein NP of the Lassa fever virus), the 4089 th and 415 th positions of SEQ ID NO.1 are terminator T5nT sequences, the 4096 th and 4129 th positions of SEQ ID NO.1 are Loxp genes, the 4130 th and 4169 th positions of SEQ ID NO.1 are promoter PE/L sequences, the 4170 th and 4889 th positions of SEQ ID NO.1 are the gene sequences of the exogenous selection marker protein EGFP, the 4890 th and 4896 th positions of SEQ ID NO.1 are terminator T5nT sequences, the 4897 th and 4930 th positions of SEQ ID NO.1 are Loxp genes, and the 4931 st and 5945 th positions of SEQ ID NO.1 are the downstream homologous arm R sequences of the TK gene.

The recombinant virus improves the safety of clinical application by knocking out strict nonessential regions of the poxvirus, can stably exist and efficiently generates the lassa fever virus empty capsid in the whole process of early and late stages; the vaccine is used for immunizing mice and rhesus monkeys, and can generate high-titer anti-Lassa fever virus antibodies to block infection of sensitive cells by different Lassa fever virus pseudoviruses.

Drawings

FIG. 1 is a schematic diagram of the structure of a transfer plasmid of the present invention.

FIG. 2 is a diagram of a recombinant vaccinia virus of the invention with or without deletion of the EGFP gene; the left panel is a recombinant vaccinia virus deleted of the EGFP gene, and the right panel is a recombinant vaccinia virus containing the EGFP gene.

FIG. 3 is an SDS-PAGE electrophoresis; wherein, the sample 1 is a vaccinia virus Tiantan strain, the sample 2 is a recombinant vaccinia virus without EGFP gene, and M is Marker.

FIG. 4 is a Western-blot detection chart; wherein, the sample 1 is a vaccinia virus Tiantan strain, and the sample 2 is a recombinant vaccinia virus without EGFP gene.

FIG. 5 is a fluorescent image of pseudoviruses in the microscale rapid fluorescence spot inhibition assay of the present invention; the left panel shows the pseudovirus after control serum neutralization, and the right panel shows the pseudovirus after recombinant virus immunized mouse serum neutralization.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Sources of cells, strains and vectors referred to in the following examples:

MRC-5(NO. CCL171) cells and Vero (NO. CCL81) cells are mainly used for preparing virus seeds of human vaccines; the primary chick embryo epithelial cells and BHK-21(NO. CCL10) cells are mainly virus seeds for preparing veterinary vaccines. MRC-5(NO.CCL171) cells, Vero (NO.CCL81) cells, BHK-21(NO.CCL10) were purchased from ATCC, and primary chick embryo epithelial cells were purchased from SPF chick embryos and prepared according to the prior art.

Recipient bacteria e.coli JM109, DH5 α were purchased from beijing holo-type gold biotechnology limited.

Vaccinia virus Tiantan strain is a product of virology research institute of Chinese disease prevention and control center and is described in non-patent document Raney.

The plasmid pBluescript was purchased from Agilent under the reference 212250.

Plasmid pVAX1 was purchased from invitrogen under the accession number V260-20.

The pVAX1-Cre plasmid was purchased from Biovector Science Lab, Inc. under the accession number 99249.

A three-plasmid lentivirus packaging system (comprising Lassa fever virus GPC nucleic acid vaccine, pLV, pHelper1.0) was purchased from Beijing English flourishing Biotech Co., Ltd., Cat. having a product number of KLV 3501.

293V Lentiviral packaging cells were purchased from Beijing Yang Sheng Biotech Co., Ltd under the trade designation C1201.

In the following examples, genetic engineering experiments involving preparation and transformation of competent cells, extraction of plasmids, recovery of DNA fragments by restriction enzyme digestion, ligation of DNA fragments, screening and identification of recombinant plasmids, PCR, and the like were carried out in accordance with the relevant section of molecular cloning, 2 nd edition. Pseudoviruses were prepared and purified according to the kit instructions.

Example 1 construction of vaccinia Virus shuttle vector/transfer plasmid pBSTKLSVLP

The structure schematic diagram of the transfer plasmid pBSTKLSVLP is shown in figure 1, the transfer plasmid takes pBluescript as a skeleton vector, and is constructed by cloning a gene component (namely, a recombinant DNA molecule), and the specific steps are as follows:

firstly, screening the vaccinia virus gene insertion region, namely determining the TK gene of the strict nonessential region in the vaccinia virus Tiantan strain

1. Preliminary screening of foreign Gene insertion region

The intergenic region is screened according to the complete gene sequence of the vaccinia virus Tiantan strain published by GenBank (GenBank number is AF095689.1), and on the basis of analysis and evaluation, the nonessential region L79784-80283 and R80841-81855(TJ2R) of the Tiantan strain are preliminarily determined, and other nonessential regions such as L138353-138861 and R139585-140607(TA35L) nucleotides can be selected as the exogenous gene insertion region.

2. Obtaining the upstream homology arm L and the downstream homology arm R of the TK gene of the poxvirus strict nonessential region gene

According to the vaccinia virus Tiantan strain whole gene sequence (GenBank number is AF095689.1) published in GenBank, after analyzing and homology comparing the thymidine kinase TK gene of the vaccinia virus Tiantan strain and its flanking sequence, according to the relationship between the length of homologous sequence and recombination efficiency, selecting two nucleotide sequences with the length more than or equal to 500bp, and adding proper restriction enzyme cutting sites to obtain the upstream homologous arm L (1 st-500 th position of SEQ ID NO. 1) and the downstream homologous arm R (4931 st-5945 th position of SEQ ID NO. 1).

II, obtaining the gene component in the transfer plasmid pBSTKLSVLP

The gene component in the whole gene synthesis transfer plasmid pBSTKLSVLP is based on the upstream and downstream homologous arms (TKL and TKR) of thymidine kinase TK gene which is strictly non-essential region gene of poxvirus, four independent exogenous gene expression cassettes which take PE/L as promoter and T5nT as terminator are inserted in the middle in sequence, and the exogenous genes respectively optimize the genes of structural protein Z, GPC and NP of the lassa fever virus and the gene of exogenous screening marker protein EGFP (wherein, both sides of the gene expression cassette of the screening marker protein EGFP also contain Loxp genes), and respectively express recombinant DNA molecules constructed by structural protein Z, GPC and NP of the lassa fever virus and the screening marker protein EGFP; the sequence of the gene component in the transfer plasmid pBSTKLSVLP is shown as SEQ ID NO.1, wherein, the 1 st to 500 th sites of SEQ ID NO.1 are the upstream homology arm L sequence of TK gene, the 501 st and 507 th sites of SEQ ID NO.1 are the reverse complement sequence of terminator T5nT sequence, the 508 nd and 807 th sites of SEQ ID NO.1 are the reverse complement sequence of the optimized lasalo-fever virus structural protein Z gene, the 808 nd and 847 th sites of SEQ ID NO.1 are the reverse complement sequence of promoter PE/L sequence, the 24164 nd 2370 th site of SEQ ID NO.1 is the promoter PE/L sequence, the 888 and 2363 th sites of SEQ ID NO.1 are the optimized lasalo-fever virus structural protein GPC gene sequence, the 2364 nd 2370 th site of SEQ ID NO.1 is the terminator T5nT sequence, the 232410 th site of SEQ ID NO.1 is the promoter/L sequence, the 2410 th site of the optimized lanalo-fever virus structural protein NP 1 st site of SEQ ID NO.1 is the terminator NP 2 st site of the optimized lasalo structural protein NP 1 (SEQ ID NO. 1) 4088 is the coding sequence of the optimized structural protein NP of the Lassa fever virus), the 4089 th position of SEQ ID NO.1 is the terminator T5nT sequence, the 4096 th position 4129 th position of SEQ ID NO.1 is the Loxp gene, the 4130 th position 4169 of SEQ ID NO.1 is the promoter PE/L sequence, the 4170 th position 4889 of SEQ ID NO.1 is the gene sequence of the exogenous selection marker protein EGFP, the 4890 th position 4896 th position of SEQ ID NO.1 is the terminator T5nT sequence, the 4897 th position 4930 of SEQ ID NO.1 is the Loxp gene, and the 4931 th position 5945 of SEQ ID NO.1 is the downstream homologous arm R sequence of the TK gene. The amino acid sequence of the protein coded by the structural protein Z gene of the lassa fever virus is shown as SEQ ID NO.2, the amino acid sequence of the protein coded by the structural protein GPC gene of the lassa fever virus is shown as SEQ ID NO.3, and the amino acid sequence of the protein coded by the structural protein NP gene of the lassa fever virus is shown as SEQ ID NO. 4.

Thirdly, cloning the gene component shown in SEQ ID NO.1 into the multiple cloning site of the plasmid pBluescript to construct pBSTKLSVLP, and determining the concentration of nucleic acid by spectrophotometry, wherein 0D260/280 is between 1.8 and 2.0, and storing at-20 ℃ for later use.

Example 2 screening and identification of recombinant vaccinia Virus

First, cell culture, digestion passage and cell counting

MRC-5(NO. CCL171) cells were cultured in a complete MEM culture medium containing 5% FCS at 37 ℃ in a 5% carbon dioxide incubator. After MRC-5 cells grow into a monolayer in a culture bottle, pouring out MEM complete culture solution in the culture bottle under the aseptic condition, adding a small amount of 0.25% pancreatin solution for washing twice, then adding a small amount of pancreatin, standing and digesting until all cells on the wall fall off, adding a certain volume of 5% FCS MEM complete culture solution in each culture bottle, blowing and beating uniformly, subpackaging in culture holes of six-hole plates or culture bottles, and culturing in a 5% carbon dioxide incubator at 37 ℃ to obtain cell suspension.

0.1m1 of cell suspension is taken, diluted properly and dropped into a blood cell counting plate, and the total number of cells in four lattices at four corners is counted according to a leucocyte counting method. When counting, only cells with intact nuclei and cytoplasm are counted, and the piled cells are counted by one cell. The total number of cells in the 4 large squares was converted to the number of cells per ml of cell suspension by the calculation formula of the erythrocyte visual counting method.

Second, vaccinia virus Tiantan strain virus titer determination

MRC-5 monolayers were prepared and plated at 3 ten thousand/m 1 cells on 96 well cell culture plates, 100. mu.l per well, and cultured until the cells grew into confluent monolayers. The culture medium was discarded, the cells were washed twice with serum-free MEM medium, and the vaccinia virus Tiantan strain was diluted 10-fold with serum-free MEM medium, and then 100. mu.1 virus dilutions were sequentially added to each well, and triplicate were performed for each gradient, while blank control was set, and 5% carbon dioxide incubator was used for 2 hours at 37 ℃. Then, 100u1 MEM complete culture medium (containing 1% low melting point agar) containing 5% FCS was added thereto, and the mixture was further cultured in a 5% carbon dioxide incubator at 37 ℃ for 96 hours to obtain a virus-proliferating solution.

Plaque formation was directly observed under an inverted fluorescence microscope for each dilution, and Plaque Formation Units (PFU) contained in each ml of the virus solution were calculated according to the formula.

Thirdly, transfection and homologous recombination

Inoculating the MRC-5 cell suspension obtained in the step one into the cell holes of the culture plate, infecting the virus liquid of the vaccinia virus Tiantan strain obtained in the step two with 0.1MOI when the cells grow to 80 percent and are fused, and infecting the virus liquid for 1-2 hours at 37 ℃ by 5 percent carbon dioxide (the culture plate is gently shaken every half hour); 100. mu.10 of pti-MEM was added to 10. mu.g of the plasmid pBSTKLSVLP prepared in example 1 and mixed to obtain a mixture A; to 100. mu.l of Opti-MEM was added 8. mu.1 liposomes, and the mixture was gently mixed and allowed to stand at room temperature for 5min to obtain mixture B. Then, the mixture B was slowly dropped into the mixture A, gently mixed, acted at room temperature for 20 minutes, added to the cells obtained by culturing in the wells of the plate, cultured at 37 ℃ for 6 hours with 5% carbon dioxide, replaced with freshly prepared MEM containing 5% FCS, cultured for further 24 hours, then the infection solution was discarded, added to a MEM medium containing 1% low-melting agarose, and after 72 hours, viral plaques were obtained, and the appearance of green fluorescence was observed under an inverted fluorescence microscope.

The vaccinia virus Tiantan strain of untransfected transfer plasmid pBSTKLSVLP was used as a negative control, and the presence of green fluorescence was also observed under an inverted fluorescence microscope.

Fluorescence detection results show that the vaccinia virus Tiantan strain transfected with the transfer plasmid pBSTKLSVLP has green fluorescence, namely, exogenous genes can be efficiently expressed, negative control has no fluorescence, and the recombinant vaccinia virus is preliminarily determined to be obtained.

Genome sequencing identification of recombinant vaccinia virus

And (3) carrying out repeated freeze thawing on the virus plaques with fluorescence, cracking cells, releasing viruses, and centrifuging at 3000-6000 rpm to remove cells or cell debris. The supernatant was extracted using the "column viral genome extraction kit" (Qiagen) for recombinant vaccinia virus genomes, see kit instructions. Sent to the company Limited of engineering bioengineering for sequencing to determine whether the integration site and the sequence are correct.

The genome sequencing detection result shows that the structural protein Z gene of the lassa fever virus, the structural protein GPC gene of the lassa fever virus, the structural protein NP gene of the lassa fever virus and the EGFP gene are integrated between the upstream and downstream homologous arms of the TK gene of the heaven altar strain of the vaccinia virus, and the recombinant vaccinia virus containing the EGFP gene is obtained.

Fifthly, transfection and EGFP gene deletion

And inoculating the MRC-5 cell suspension obtained in the step one into the cell wells of the culture plate, infecting the recombinant vaccinia virus obtained in the step four with 0.1MOI when the cells grow to 80 percent of fusion, infecting the recombinant vaccinia virus with 5 percent carbon dioxide at 37 ℃ for 1-2 hours (slightly shaking the culture plate every half hour), and then transfecting the cells with pVAX1-Cre plasmid. Adding 100 mu 10 pti-MEM into 10 mu g of pVAX1-Cre plasmid DNA, and mixing uniformly; to 100. mu.l of Opt i-MEM, 8. mu.1 liposomes were added, gently mixed, and allowed to stand at room temperature for 5 min. Then, the latter was slowly added dropwise to the former, gently mixed, allowed to act at room temperature for 20 minutes, added to the wells, cultured at 37 ℃ for 6 hours in 5% carbon dioxide, replaced with freshly prepared MEM containing 5% FCS, cultured for 24 hours, then the infection solution was discarded and added to a 1% MEM medium containing low melting point agarose, and after 72 hours, viral plaques were obtained, and the plaques were observed under an inverted fluorescence microscope for the presence of green fluorescence.

The fluorescence detection result is shown in fig. 2, and the result shows that compared with the recombinant vaccinia virus containing the EGFP gene obtained in the step four, no green fluorescence appears, and the recombinant vaccinia virus with the EGFP gene deleted is obtained through preliminary determination.

Sixth, identification of recombinant vaccinia Virus

And harvesting the virus plaques without fluorescence, repeatedly freezing and thawing, cracking cells, releasing viruses, and centrifuging at 3000-6000 rpm to remove cells or cell debris. The supernatant was extracted using the "column viral genome extraction kit" (Qiagen) for recombinant vaccinia virus genomes, see kit instructions. Sequencing by the company of engineering and biological engineering, and determining whether the EGFP gene is deleted and whether the rest sequences are correct.

The genome sequencing detection result shows that the EGFP gene is deleted without influencing other genes, and the recombinant vaccinia virus without the EGFP gene is obtained.

Example 4 recombinant vaccinia Virus-expressed Lassa fever structural protein and empty capsid Assembly assay

SDS-PAGE electrophoresis

Preparing a single-layer Vero (NO. CCL81) cell, discarding the supernatant, adding 1% of the recombinant vaccinia virus which is obtained in example 3 and does not contain EGFP gene, sensing the temperature at 37 ℃ for 2 hours, fully adsorbing, adding a 199 culture medium containing 6-8% fetal calf serum, continuously culturing until the lesion is obvious after 14 hours, cleaning the cell by PBS, adding a serum-free culture medium, continuously culturing for 24 hours, repeatedly freezing and thawing for 3 times, harvesting the Vero cell and the supernatant infected with the recombinant virus, taking the supernatant 90 mu 1, adding 4 xSDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) sample loading buffer solution, uniformly mixing, boiling for 10 minutes, preparing an electrophoresis sample, carrying out 12% polyacrylamide gel electrophoresis, dyeing for 1 hour after the completion, decoloring overnight, and observing a map.

The recombinant vaccinia virus not containing the EGFP gene was replaced with a vaccinia virus Tiantan strain, and the test was performed in the same manner as a control.

The electrophoresis results are shown in FIG. 3, and the results show that the recombinant vaccinia virus strain has bands with sizes corresponding to GPC, NP and Z proteins at positions near 75Kd, 60Kd and 12Kd, and no corresponding band is observed in the vaccinia virus Tiantan strain.

Second, Western-blot detection

Transferring a PVDF membrane to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis gel of a recombinant vaccinia virus without EGFP (enhanced Green fluorescent protein) gene at 100V for 45 minutes, sealing with 5% skim milk PBS overnight, taking the PBS as a diluent, combining the recovered serum of a Lassa fever patient at the temperature of 1:50 for 1 hour at the temperature of 37 ℃, fully washing with the PBS, combining a secondary antibody HRP-goat anti-human IgG (diluted at the temperature of 1: 1000) at the temperature of 37 ℃ for 1 hour, performing DAB (digital audio broadcasting) color development, and observing a map.

Western-blot detection is shown in FIG. 4, and the results show that: the recombinant vaccinia virus (sample 2) has bands with sizes equivalent to those of GPC, NP and Z proteins at positions near 75Kd, 60Kd and 12Kd, while the vaccinia virus Tiantan strain (sample 1) has no corresponding band, and the results show that the recombinant vaccinia virus can effectively recognize the recovered serum of patients with Lassa fever, and the vaccinia virus Tiantan strain cannot recognize the recovered serum of patients with Lassa fever.

Third, detecting empty capsid by electron microscope

Washing the Vero cells infected with the recombinant vaccinia virus in the step I for 3 times by PBS, reserving about 1mL of PBS, repeatedly freezing and thawing for 3 times, collecting 1mL of cell culture, centrifuging for 1min at 12,000rpm at 4 ℃, taking about lmL of supernatant, adding 10 mu L of recovery serum, acting for 30min at 37 ℃, rapidly placing for balancing for 1 h at 4 ℃, centrifuging for 30min at 12,000rpm at 4 ℃, discarding 900ul of supernatant, reserving 100u of 1 of supernatant for dissolving and precipitating as an electron microscope sample, carrying out phosphotungstic acid negative staining, and observing under a transmission electron environment.

The detection of an immunoelectron microscope shows that: virus-like particles with the length of about 7-10nm can be observed in a recombinant vaccinia virus sample without EGFP gene, and corresponding particles cannot be observed in a control sample of a vaccinia virus Tiantan strain.

The detection results support that the recombinant vaccinia virus without EGFP gene not only expresses structural protein of the lassa fever virus, but also can be assembled into empty capsids, so that the recombinant vaccinia virus which expresses and assembles the lassa fever virus empty capsids in host cells is successfully screened by the construction method of the recombinant vaccinia virus.

Example 5 preparation and detection of Lassa fever Virus pseudoVirus

Preparation and detection of Lassa fever virus pseudovirus

Transfecting a 293V lentivirus packaging cell with a Lassa fever virus GPC nucleic acid vaccine, pLV and pHelper1.0 three plasmids according to the three-plasmid lentivirus packaging system operation instruction, packaging Lassa fever virus pseudoviruses to obtain Lassa fever virus pseudoviruses, establishing a pseudovirus library, measuring the titer by using Vero cells, wherein the pseudovirus titer is 1 multiplied by 10^5-7FFU/ml。

Second, immunizing healthy animals

Adopting a subcutaneous immunization strategy to immunize 5 quarantine qualified SPF grade Balb/C mice, each of which contains 10 mice^3-5The recombinant vaccinia virus not containing the EGFP gene obtained in PFU example 3 is collected at intervals of 21-28 days, and serum is collected and stored below-70 ℃ for later use.

Sera from non-immunized, quarantined, qualified, SPF grade Balb/C mice were used as controls.

Adopting subcutaneous immunization strategy to immunize 5 quarantine-qualified rhesus monkeys, each 10^4-6The recombinant vaccinia virus not containing the EGFP gene obtained in PFU example 3 is collected at intervals of 21-28 days, and serum is collected and stored below-70 ℃ for later use.

Sera from naive rhesus monkeys were used as controls.

Thirdly, measuring the titer of the neutralizing antibody of the lassa fever virus

A micro rapid fluorescence focus inhibition test method is adopted to measure the titer of the specific immunoglobulin neutralizing antibody of the anti-tension sand fever virus, and the specific method is as follows:

(1) a96-well plate is transversely used, each plate can be used for preparing 6 parts of serum to be detected, and the serum to be detected is serially diluted. Adding 50 mul of diluent into each hole of a 96-hole plate; A1-A12, adding 50 mul of corresponding sample into each hole, mixing uniformly from the line A, sucking 50 mul to the line B, mixing uniformly, diluting to the line G, sucking 50 mul of antibody in the line G, and discarding; adding H1-H6 into 50 mul of corresponding serum sample to be detected, and mixing uniformly; the cell control wells were supplemented with 50. mu.l of antibody diluent and mixed well.

(2) Diluting Lassa fever virus pseudovirus to 100FFU/0.05ml, and dripping 50 μ l of the vertical suspension into each well of a 96-well plate (except serum and cell control); taking another 1.5ml of 25000FFU/0.05ml of virus in a small centrifugal tube, temporarily storing at 4 ℃ until the virus titer is detected again;

(3) gently and uniformly mixing the cell culture plate, and neutralizing for 1 hour at 37 ℃;

(4) virus titer retesting: taking 4 small tubes, adding 0.9ml of diluent into each tube, sucking 0.1ml of 100FFU/0.05ml of lassa fever virus pseudovirus, adding into the 1 st tube, uniformly mixing to obtain 10FFU/0.05ml, and sequentially diluting by 10 times to 1FFU/0.05 ml; taking another 96-well plate, adding 0.1ml of each diluted virus into each well, and making 8 multiple wells; meanwhile, 4 holes are reserved as cell control holes, and 0.1ml of virus diluent is added into each hole;

(5) digesting the cells with digestive juice to obtain 2 × 10⁵Adding 0.1ml cell suspension into each well, mixing, placing at 35 deg.C and 5% CO₂Incubating and culturing in an incubator; counting the fluorescence focus value by a fluorescence microscope, recording the virus titration result, taking the reciprocal of the highest dilution of the serum inhibiting 50% of the fluorescence focus as the end point titer, and judging the final result after 2 days.

The basis for judging the result is as follows: when 1 hole of 2 holes of the serum with the highest dilution generates a fluorescence focus, the other hole does not generate the fluorescence focus, and the reciprocal of the dilution is the titer of the neutralizing antibody of the serum specimen; when a fluorescence focus appears in the high dilution 2 hole and no fluorescence focus appears in the adjacent low dilution 2 hole, the reciprocal of the average dilution of the two is the titer of the neutralizing antibody of the serum specimen; when 1-hole fluorescence focus appears in two adjacent dilutions of serum and no fluorescence focus appears in the other 1-hole, the reciprocal of the average dilution of the two dilutions is the titer of the neutralizing antibody of the serum specimen. Note that: if the virus back-drop result is not within the range of 32-320 FFU/0.05ml, the experiment is invalid, and the experiment is repeated.

The results are shown in fig. 5, and the antibody titers of the obtained mouse serum and the lassa fever virus pseudovirus are 1: 512. 1: 256. 1: 128. 1: 512 and 1: 256.

the antibody titers of the rhesus monkey serum neutralizing the lassa fever virus pseudovirus are respectively 1: 256. 1: 128. 1: 512. 1: 256 and 1: 528.

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 embodiments, it will be appreciated that the invention can 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> military medical research institute of military science institute of people's liberation force of China

<120> recombinant virus for expressing Lassa fever virus empty capsid and preparation method thereof

<130>GNCFY200076

<160>4

<170>PatentIn version 3.5

<210>1

<211>5945

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ataaagattg tgcaatgctt ttgcgatcaa taaatggatc acaaccagta tctcttaacg 60

atgttcttcg cagatgatga ttcatttttt aagtatttgg ctagtcaaga tgatgaatct 120

tcattatctg atatattgca aatcactcaa tatttagact ttctgttatt attattgatc 180

caatcaaaaa ataaattaga agccgtgggt cattgttatc aatctctttc agaggaatac 240

agacaattga caaaattcac agactctcaa gattttataa aactgtttaa caaggtccct 300

attgttacag atggaagggt caaacttaat aaaggatatt tgttcgactt tgtgattagt 360

ttgatgcgat tcaaaaaaga atcctctcta gctaccaccg caatagatcc tattagatac 420

atagatcctc gtcgcgaaat cgcattttct aacgtgatgg atatattaaa gtcgaataaa 480

gtgaacaata attaattctt acaaaaatta gggtgtgtat ggtgggggat tggtgatgct 540

ctccctggtg atttgtggtg ctgttggagc ggctgatggt ctcagttttg tggggagagg 600

catcttgcaa atgggacacc tgttgctgac agacaggagt aaagtaaggc agttgaggca 660

taagtaatgg ttgttgcact ccaccagccc tctgttctca aaccagcaac tcttgcagaa 720

ctgggggcct agatgggatg catctggtag cagggttggt ctggggtgct ctggtttggg 780

tggggacctg gtctgcttgt tgcccattat ttatattcca aaaaaaaaaa ataaaatttc 840

aatttttaaa aattgaaatt ttattttttt tttttggaat ataaataatg gggcagatta 900

ttacattctt tcaagaagtg ccacatgtaa tagaggaagt catgaacatt gtgctaattg 960

cgctttctct attggcaatc ttgaagggct tgtataacat cgctacatgt gggattattg 1020

gattggttgc ctttttattc ttgtgtggca agtcttgttc cctaaccctt aaagggggat 1080

atgagctgca aaccttagaa ttaaatatgg agaccctaaa catgaccatg cccttatcat 1140

gcaccaagaa cagcagtcat cattacataa gagtgggcaa tgagactggc ttagagctga 1200

ccttaacaaa tacaagtatc atcaatcaca aattttgtaa cctttctgat gcacataaaa 1260

agaatcttta tgaccatgct ttaatgagta tcatctcaac ctttcattta tccattccta 1320

actttaatca gtatgaagca atgagttgtg acttcaatgg ggggaagata agtgttcagt 1380

acaaccttag ccacgcttat gctgtagatg cagctaacca ttgtgggact attgccaatg 1440

gcgttcttca gactttcatg aggatggctt ggggtggcag ttacatagcc cttgattccg 1500

gacgtgggaa atgggattgt ataatgacca gttatcaata cttaatcatc cagaacacaa 1560

cctgggaaga tcattgtcaa ttttccagac catccccaat tggatatctt gggctcctct 1620

cacagagaac tagagacata tacattagta gaagactact aggaacattt acttggacac 1680

tttctgactc agaaggaaat gagacaccag gaggttactg tctcactaga tggatgttga 1740

ttgaggctga actaaagtgt ttcgggaata cagctgtagc taagtgctgt aatgagaagc 1800

atgatgagga attttgtgac atgctgaggc tgtttgactt caacaaacaa gccattcaaa 1860

ggttgaaagc tgaagcacaa atgagcattc agttgatcaa caaagcagta aatgctttga 1920

taaatgacca acttataatg aagaaccatc tacgggacat catgggaatt ccatactgta 1980

attacagcaa gtattggtac ctcaaccaca caactactgg gagaacatca ctgcccaaat 2040

gttggcttgt atcaaatggt tcatacttga acgagaccca cttttctgat gacatcgaac 2100

agcaagccga caacatgatc acagagatgc tacaaaagga atacatggac aggcaaggaa 2160

aaacaccttt gggcctggtt gacttgtttg tgtttagcac aagtttttat ctgattagca 2220

ttttccttca tttagtcaaa ataccgaccc atagacacat tgtgggtaaa ccgtgtccca 2280

agcctcacag gctgaaccgc atgggcattt gctcttgtgg tttgtacaag caacctggcg 2340

tccctgttaa atggaagaga tagtttttgt aaaaattgaa attttatttt ttttttttgg 2400

aatataaata atggacacag tctttgagga gagaattatc gggctactgt tccaacatca 2460

agctgcaggt ggtgaaggat gcccaagctc ttctacatgg acttgatttt tcagaggtca 2520

gcaatgtcca acggctgatg cgcaagcaga ggagggatga tggtgatcta aaacgactca 2580

gggacctaaa tcaagcggtc aacaatcttg ttgaattaaa atcaactcaa caaaagagta 2640

tactgagagt tgggactcta acctcagatg acttattaat cttagccgct gatctagaga 2700

agttaaagtc aaaggtgatc agaacagaaa ggccattaag tgcaggtgtc tatatgggca 2760

acctaagctc acagcaactt gaccaaagaa gagctctcct gaatatgataggaatgagtg 2820

gtggtaatca aggggctcgg gctgggagag atggagtggt gagagtttgg gatgtgaaaa 2880

atgcagagct gcttaacaat cagtttggga caatgccaag tctaactcta gcatgtttga 2940

ccaaacaagg gcaagtggat ttgaatgatg ctgttcaggc tttgacagat ttgggattga 3000

tttacaccgc aaaataccct aattcatctg atttggacag gctggcccag agccatccga 3060

tattgaacat gattgatact aagaagagct cccttaacat ctctggttac aattttagtt 3120

taggagccgc cgtcaaggct ggggcctgca tgcttgatgg aggcaatatg ctagagacta 3180

taaaggtctc acctcaaacc atggatggca tcttgaagtc aatcttgaaa gtcaagagaa 3240

gcctgggaat gttcatctca gatacgccag gtgaaaggaa cccttatgag aacatacttt 3300

acaagatctg tctttccggt gacggctggc cctacatagc ctcaaggact tccattgtgg 3360

gtagagcttg ggagaatact actgtagatc tcgagtcaga tggaaaacca caaaaggtgg 3420

gaactgctgg gtccaacaag tctttgcagt cagcggggtt tccaacaggg ctgacttatt 3480

ctcagttgat gacacttaaa gactcaatga tgcagcttga tccaagtgcc aaaacctgga 3540

tagacattga aggtcgtcca gaagaccctg tggaaatagc tctttaccaa cctatgtcgg 3600

gctgctatat acatttcttc agagaaccaa cagacttaaa acagttcaag caagatgcca 3660

aatattctca tgggattgat gtaacagatc ttttttccac acaacccggg ctgacaagtg 3720

ctgtcataga ggcacttccc cgcaatatgg tgttgacctg ccaggggtca gatgacatca 3780

aaaagctgtt ggagtctcaa gggagaagag acatcaaatt gattgatatt tctttgagca 3840

aagcagattc aaggaagttt gaaaatgctg tgtgggacca atacaaagac ttgtgtcaca 3900

tgcacacggg agtagtcgtt gagaagaaaa agaggggtgg aaaagaggaa atcaccccac 3960

attgcgcttt actggactgc atcatgtttg atgcagctac tactggtagt ttgaacatca 4020

caacactgag ggcagtactt cccagagaca tggtcttcag gaccagcacc cctaaagtcg 4080

ttctgtaatt tttgtataac ttcgtataat gtatgctata cgaagttata aaaattgaaa 4140

ttttattttt tttttttgga atataaataa tggtgagcaa gggcgaggag ctgttcaccg 4200

gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt 4260

ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 4320

ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt 4380

gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg 4440

aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg 4500

ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact 4560

tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg 4620

tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca 4680

acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg 4740

acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag 4800

accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca 4860

ctctcggcat ggacgagctg tacaagtaat ttttgtataa cttcgtataa tgtatgctat 4920

acgaagttat atctaaaaaa ctaaaaataa acattgatta aattttaata taatacttaa 4980

aaatggatgt tgtgtcgtta gataaaccgt ttatgtattt tgaggaaatt gataatgagt 5040

tagattacga accagaaagt gcaaatgagg tcgcaaaaaa actgccgtat caaggacagt 5100

taaaactatt actaggagaa ttattttttc ttagtaagtt acagcgacac ggtatattag 5160

atggtgccac cgtagtgtat ataggatctg ctcccggtac acatatacgt tatttgagag 5220

atcatttcta taatttagga gtgatcatca aatggatgct aattgacggc cgccatcatg 5280

atcctatttt atatggattg cgtgatgtga ctctagtgac tcggttcgtt gatgaggaat 5340

atctacgatc catcaaaaaa caactgcatc cttctaagat tattttaatt tctgatgtga 5400

gatccaaacg aggaggaaat gaacctagta cggcggattt actaagtaat tacgctctac 5460

aaaatgtcat gattagtatt ttataccccg tggcatctag tcttaaatgg agatgcccgt 5520

ttccagatca atggatcaag gacttttata tcccacacgg taataaaatg ttacaacctt 5580

ttgctccttc atattcagct gaaatgagat tattaagtat ttataccggt gagaacatga 5640

gactgactag agttaccaaa tcagacgctg taaattatga aaaaaagatg tactacctta 5700

ataagatcgt ccgtaacaaa gtagttgtta actttgatta tcctaatcag gaatatgact 5760

attttcacat gtactttatg ctgaggaccg tgtactgcaa taaaacattt cctactacta 5820

aagcaaaggt actatttcta caacaatcta tatttcgttt cttaaatatt ccaacaacat 5880

caactgaaaa agttagtcat gaaccaatac aacgtaaagt atctagcaaa gattctatgt 5940

ctaaa5945

<210>2

<211>99

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Met Gly Asn Lys Gln Thr Arg Ser Pro Pro Lys Pro Glu His Pro Arg

1 5 10 15

Pro Thr Leu Leu Pro Asp Ala Ser His Leu Gly Pro Gln Phe Cys Lys

20 25 30

Ser Cys Trp Phe Glu Asn Arg Gly Leu Val Glu Cys Asn Asn His Tyr

35 40 45

Leu Cys Leu Asn Cys Leu Thr Leu Leu Leu Ser Val Ser Asn Arg Cys

50 55 60

Pro Ile Cys Lys Met Pro Leu Pro Thr Lys Leu Arg Pro Ser Ala Ala

65 70 75 80

Pro Thr Ala Pro Gln Ile Thr Arg Glu Ser Ile Thr Asn Pro Pro Pro

85 90 95

Tyr Thr Pro

<210>3

<211>491

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Met Gly Gln Ile Ile Thr Phe Phe Gln Glu Val Pro His Val Ile Glu

1 5 10 15

Glu Val Met Asn Ile Val Leu Ile Ala Leu Ser Leu Leu Ala Ile Leu

20 25 30

Lys Gly Leu Tyr Asn Ile Ala Thr Cys Gly Ile Ile Gly Leu Val Ala

35 40 45

Phe Leu Phe Leu Cys Gly Lys Ser Cys Ser Leu Thr Leu Lys Gly Gly

50 55 60

Tyr Glu Leu Gln Thr Leu Glu Leu Asn Met Glu Thr Leu Asn Met Thr

65 70 75 80

Met Pro Leu Ser Cys Thr Lys Asn Ser Ser His His Tyr Ile Arg Val

85 90 95

Gly Asn Glu Thr Gly Leu Glu Leu Thr Leu Thr Asn Thr Ser Ile Ile

100 105 110

Asn His Lys Phe Cys Asn Leu Ser Asp Ala His Lys Lys Asn Leu Tyr

115 120 125

Asp His Ala Leu Met Ser Ile Ile Ser Thr Phe His Leu Ser Ile Pro

130 135 140

Asn Phe Asn Gln Tyr Glu Ala Met Ser Cys Asp Phe Asn Gly Gly Lys

145 150 155 160

Ile Ser Val Gln Tyr Asn Leu Ser His Ala Tyr Ala Val Asp Ala Ala

165 170 175

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

180 185 190

Met Ala Trp Gly Gly Ser Tyr Ile Ala Leu Asp Ser Gly Arg Gly Lys

195 200 205

Trp Asp Cys Ile Met Thr Ser Tyr Gln Tyr Leu Ile Ile Gln Asn Thr

210 215 220

Thr Trp Glu Asp His Cys Gln Phe Ser Arg Pro Ser Pro Ile Gly Tyr

225 230 235 240

Leu Gly Leu Leu Ser Gln Arg Thr Arg Asp Ile Tyr Ile Ser Arg Arg

245 250 255

Leu Leu Gly Thr Phe Thr Trp Thr Leu Ser Asp Ser Glu Gly Asn Glu

260 265 270

Thr Pro Gly Gly Tyr Cys Leu Thr Arg Trp Met Leu Ile Glu Ala Glu

275 280 285

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

290 295 300

His Asp Glu Glu Phe Cys Asp Met Leu Arg Leu Phe Asp Phe Asn Lys

305 310 315 320

Gln Ala Ile Gln Arg Leu Lys Ala Glu Ala Gln Met Ser Ile Gln Leu

325 330 335

Ile Asn Lys Ala Val Asn Ala Leu Ile Asn Asp Gln Leu Ile Met Lys

340 345 350

Asn His Leu Arg Asp Ile Met Gly Ile Pro Tyr Cys Asn Tyr Ser Lys

355 360 365

Tyr Trp Tyr Leu Asn His Thr Thr Thr Gly Arg Thr Ser Leu Pro Lys

370 375 380

Cys Trp Leu Val Ser Asn Gly Ser Tyr Leu Asn Glu Thr His Phe Ser

385 390 395 400

Asp Asp Ile Glu Gln Gln Ala Asp Asn Met Ile Thr Glu Met Leu Gln

405 410 415

Lys Glu Tyr Met Asp Arg Gln Gly Lys Thr Pro Leu Gly Leu Val Asp

420 425 430

Leu Phe Val Phe Ser Thr Ser Phe Tyr Leu Ile Ser Ile Phe Leu His

435 440 445

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

450 455 460

Lys Pro His Arg Leu Asn Arg Met Gly Ile Cys Ser Cys Gly Leu Tyr

465 470 475 480

Lys Gln Pro Gly Val Pro Val Lys Trp Lys Arg

485 490

<210>4

<211>558

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Trp Thr Gln Ser Leu Arg Arg Glu Leu Ser Gly Tyr Cys Ser Asn Ile

1 5 10 15

Lys Leu Gln Val Val Lys Asp Ala Gln Ala Leu Leu His Gly Leu Asp

20 25 30

Phe Ser Glu Val Ser Asn Val Gln Arg Leu Met Arg Lys Gln Arg Arg

35 40 45

Asp Asp Gly Asp Leu Lys Arg Leu Arg Asp Leu Asn Gln Ala Val Asn

50 55 60

Asn Leu Val Glu Leu Lys Ser Thr Gln Gln Lys Ser Ile Leu Arg Val

65 70 75 80

Gly Thr Leu Thr Ser Asp Asp Leu Leu Ile Leu Ala Ala Asp Leu Glu

85 90 95

Lys Leu Lys Ser Lys Val Ile Arg Thr Glu Arg Pro Leu Ser Ala Gly

100 105 110

Val Tyr Met Gly Asn Leu Ser Ser Gln Gln Leu Asp Gln Arg Arg Ala

115 120 125

Leu Leu Asn Met Ile Gly Met Ser Gly Gly Asn Gln Gly Ala Arg Ala

130 135 140

Gly Arg Asp Gly Val Val Arg Val Trp Asp Val Lys Asn Ala Glu Leu

145 150 155 160

Leu Asn Asn Gln Phe Gly Thr Met Pro Ser Leu Thr Leu Ala Cys Leu

165 170 175

Thr Lys Gln Gly Gln Val Asp Leu Asn Asp Ala Val Gln Ala Leu Thr

180 185 190

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

195 200 205

Asp Arg Leu Ala Gln Ser His Pro Ile Leu Asn Met Ile Asp Thr Lys

210 215 220

Lys Ser Ser Leu Asn Ile Ser Gly Tyr Asn Phe Ser Leu Gly Ala Ala

225 230 235 240

Val Lys Ala Gly Ala Cys Met Leu Asp Gly Gly Asn Met Leu Glu Thr

245 250 255

Ile Lys Val Ser Pro Gln Thr Met Asp Gly Ile Leu Lys Ser Ile Leu

260 265 270

Lys Val Lys Arg Ser Leu Gly Met Phe Ile Ser Asp Thr Pro Gly Glu

275 280 285

Arg Asn Pro Tyr Glu Asn Ile Leu Tyr Lys Ile Cys Leu Ser Gly Asp

290 295 300

Gly Trp Pro Tyr Ile Ala Ser Arg Thr Ser Ile Val Gly Arg Ala Trp

305 310 315 320

Glu Asn Thr Thr Val Asp Leu Glu Ser Asp Gly Lys Pro Gln Lys Val

325 330 335

Gly Thr Ala Gly Ser Asn Lys Ser Leu Gln Ser Ala Gly Phe Pro Thr

340 345 350

Gly Leu Thr Tyr Ser Gln Leu Met Thr Leu Lys Asp Ser Met Met Gln

355 360 365

Leu Asp Pro Ser Ala Lys Thr Trp Ile Asp Ile Glu Gly Arg Pro Glu

370 375 380

Asp Pro Val Glu Ile Ala Leu Tyr Gln Pro Met Ser Gly Cys Tyr Ile

385 390 395 400

His Phe Phe Arg Glu Pro Thr Asp Leu Lys Gln Phe Lys Gln Asp Ala

405 410 415

Lys Tyr Ser His Gly Ile Asp Val Thr Asp Leu Phe Ser Thr Gln Pro

420 425 430

Gly Leu Thr Ser Ala Val Ile Glu Ala Leu Pro Arg Asn Met Val Leu

435 440 445

Thr Cys Gln Gly Ser Asp Asp Ile Lys Lys Leu Leu Glu Ser Gln Gly

450 455 460

Arg Arg Asp Ile Lys Leu Ile Asp Ile Ser Leu Ser Lys Ala Asp Ser

465 470 475 480

Arg Lys Phe Glu Asn Ala Val Trp Asp Gln Tyr Lys Asp Leu Cys His

485 490 495

Met His Thr Gly Val Val Val Glu Lys Lys Lys Arg Gly Gly Lys Glu

500 505 510

Glu Ile Thr Pro His Cys Ala Leu Leu Asp Cys Ile Met Phe Asp Ala

515 520 525

Ala Thr Thr Gly Ser Leu Asn Ile Thr Thr Leu Arg Ala Val Leu Pro

530 535 540

Arg Asp Met Val Phe Arg Thr Ser Thr Pro Lys Val Val Leu

545 550 555

Claims

1. A method for constructing a recombinant virus, comprising: the construction method comprises the following steps:

2. The construction method according to claim 1, characterized in that: the amino acid sequence of the protein coded by the structural protein Z gene of the lassa fever virus is shown as SEQ ID NO.2, the amino acid sequence of the protein coded by the structural protein GPC gene of the lassa fever virus is shown as SEQ ID NO.3, and the amino acid sequence of the protein coded by the structural protein NP gene of the lassa fever virus is shown as SEQ ID NO. 4.

3. The construction method according to claim 1 or 2, characterized in that: the coding sequence of the structural protein Z gene of the lassa heat virus is the reverse complementary sequence of the 508 th and 807 th positions of SEQ ID NO.1, the coding sequence of the structural protein GPC gene of the lassa heat virus is the 888-2363 th positions of SEQ ID NO.1 and the coding sequence of the structural protein NP gene of the lassa heat virus is the 2412 th and 4088 th positions of SEQ ID NO. 1.

4. The construction method according to any one of claims 1 to 3, wherein: the recombinant DNA molecule further comprises two loxp sites and a selectable marker protein gene located between the two loxp sites.

5. The construction method according to claim 4, wherein: the construction method also comprises the step of co-transfecting host cells with the pVAX1-Cre plasmid and the recombinant vaccinia virus to obtain the recombinant virus without the selection marker protein gene.

6. The construction method according to any one of claims 1 to 5, wherein: the gene of the strict nonessential region of the poxvirus is thymidine kinase TK gene.

7. A recombinant virus constructed by the method according to any one of claims 1 to 6.

8. Recombinant virus-like particle produced by the recombinant virus of claim 7.

9. The use of the recombinant virus-like particle of claim 8 in any one of the following:

1) the application in preparing Lassa fever vaccine;

2) the application in preparing the product for preventing and/or treating lassa fever;

3) the use thereof for the preparation of a product for the prophylaxis and/or treatment of diseases caused by poxviruses.

10. A transfer plasmid, characterized by: the transfer plasmid contains recombinant DNA molecules which are constructed on the basis of upstream and downstream homology arms of the genes of the non-essential regions strict by the poxvirus, and the recombinant DNA molecules comprise a Lassa fever virus structural protein Z gene, a Lassa fever virus structural protein GPC gene and a Lassa fever virus structural protein NP gene.