CN110592108B - Infectious recombinant cloning method for II-type VII-type epidemic NDV strain DHN3 - Google Patents

Abstract

The invention belongs to the technical field of biological products for livestock, and discloses a recombinant cloning method for a II-type VII epidemic NDV strain DHN3, which comprises the following steps: step 1: constructing three types of helper plasmids, wherein target fragments on the helper plasmids are NP gene, P gene and L gene respectively; step 2: constructing a DHN3 whole genome expression vector; the DHN3 whole genome obtained by artificial recombination is recombined into a vector plasmid to obtain a whole genome expression vector; the whole genome sequence of DHN3 is shown in a sequence table SEQ ID NO 1; and step 3: co-transfecting the three auxiliary plasmids and a DHN3 whole genome expression vector to a BHK-21 cell to obtain virus liquid containing recombinant virus rDNN 3; the NP gene is shown in a sequence table SEQ ID NO: the 1 middle position is 1-1591 nt; the P gene is shown in a sequence table SEQ ID NO: the position in the 1 is 1925 and 3109 nt; the L gene is shown in a sequence table SEQ ID NO: the 1 position is 8166 + 15192 nt. The method can successfully clone the recombinant virus which is completely the same as the original virus strain and has no mutation.

Description

Infectious recombinant cloning method for II-type VII-type epidemic NDV strain DHN3

Technical Field

The invention relates to the technical field of veterinary biological products, in particular to an infectious recombinant cloning method aiming at a II-type VII epidemic NDV strain DHN 3.

Background

Newcastle disease is a highly contagious and lethal disease caused by Newcastle Disease Virus (NDV) that mainly attacks chickens, turkeys, wild birds and ornamental birds, commonly known as fowl plague. Belongs to highly contact, acute and severe infectious diseases. Humans occasionally become infected, manifesting as conjunctivitis. The world health Organization (OIE) lists the infectious diseases as the infectious diseases which need to be reported, and the department of agriculture in China also lists the infectious diseases as a type of animal epidemic diseases which need to be reported. Due to the rapid propagation speed and the 100% incidence and fatality rate, once the spread seriously harms the poultry industry, immeasurable loss is caused.

The prevention of newcastle disease is a key measure in addition to rational feeding. In addition to establishing a well-established and correct immunization program, the provision of an excellent broad-spectrum, highly effective and inexpensive vaccine is fundamental to the prevention of the disease.

Newcastle disease vaccines are divided into inactivated and live vaccines. The live seedlings mainly include line I, line II (B1 strain), line III (F strain) and line IV (Lasota strain). Wherein the IV-series live vaccine (Lasota strain) is an excellent low-toxicity vaccine widely applied at home and abroad at present, has higher toxicity and immunity than the II-series vaccine, and has good safety. With the application of cloning technology, a new generation of cloned seedlings is gradually coming into the market. The new generation of attenuated live vaccine favored by the market at present mainly comprises a Newcastle disease clone live vaccine C/30 developed by the Netherlands and an IV line optimized clone vaccine developed by the American general knowledge. In addition, the live vaccine (I line clone vaccine) of the moderate-virulence clone strain in the Newcastle disease also has the advantages of mild virulence and strong safety similar to the common I line vaccine.

According to epidemiological investigations, the epidemic genotype of NDV varies with time and geographical environment. In the last 20-50 years, the fluid I-IV type was predominant, and in the 70 th year, the V type was first found to be predominant in south America and middle America so as to be in Europe. Type VI appeared in the 80 s and prevailed in the middle east, asia and europe. Type VII began to spread in 85 years and, with type VIII spreading in many countries of the world and in the 90 s, resulted in a pandemic in asia, africa and the middle east. The V-VIII types are all strong toxins. Types IX and X have been limited to local sporadic states. Because vaccines against type IV are widely used, type IV NDV has been well controlled. However, vaccines against other genotypes are still lacking and tend to be popular. Therefore, the reverse genetic cloning technology is utilized to rapidly develop the attenuated vaccine which is high-efficiency, broad-spectrum, safe and even multivalent aiming at the real-time epidemic strains. In addition, quarantine is made more difficult by the widespread use of vaccines. Because it is impossible to discriminate whether the infected chicken carries a vaccine strain or a wild strain. An artificial label can be introduced in a recombination mode, so that the artificial label can be effectively identified with wild viruses.

Newcastle Disease Virus (NDV) belongs to the order Mononegales, Paramyxoviridae. The virus has a double lipid layer envelope lined with a layer of M protein. The outer membrane is coated with fiber-like glycoproteins (HN and F) to give a panicle-like appearance. The capsule contains a long helical nucleocapsid composed of a capsid protein and a minus-strand RNA. Newcastle disease virus has 6 groups of genes encoding 6 viral proteins, i.e., hemagglutinin and neuraminidase active (HN) glycoprotein, fusion functional (F) glycoprotein, non-glycosylated inner membrane protein (M), Nucleocapsid Protein (NP), phosphoprotein (P) and high molecular weight protein (L), respectively. The total length of the NDV gene is 15186nt to 15198 nt.

It has been shown that the cloning of NDV cDNA by artificial mutation, replacement, or insertion of foreign sequences does not prevent the replication, assembly and release of the virus. cDNA clones of NDV have been used for basic research and vaccine development. The cDNA clone of the NDV can be used as a vector to express antigen proteins of other pathogenic bacteria so as to obtain the multivalent vaccine for resisting various pathogenic bacteria. The weak toxicity of the Latasa strain is changed into strong toxicity by base mutation of F gene cleavage points by Peeters and the like in 1999 (ref 1); in 2004 Huang et al exchanged the HN genes of the virulent and attenuated strains to obtain a new strain with different virulence (ref 2). In 2002, Mebastson et al successfully obtained a hybrid virus (ref3) which resists both NDV and hepatitis virus by replacing the NP protein dominant epitope of NDV with the S2 glycoprotein epitope gene of hepatitis virus; in 2006, Man et al inserted the HA gene of H7 avian influenza virus between the P-M genes of NDV B1 strain and succeeded in obtaining a hybrid virus (ref4) that is resistant to both NDV and H7 avian influenza viruses. Recently Abzeid et al succeeded in assembling the currently circulating IBV S protein in egyptian into recombinant NDV, resulting in a hybrid virus that is immune protected against both the original NDV and the corresponding IBV. (ref5) Chinese scholars also use LaSota strain as carrier to obtain multiple bivalent hybrid viruses. Such as the hybrid virus resistant to both NDV and IBDV5 (ref 6); hybrid virus resistant to both NDV and H5N1 (ref 7); hybrid virus resistant to both NDV and Mycoplasma gallisepticum TM1 virus (ref 8). Because NDV has only one serotype, its genetic properties are relatively stable. Although infectious cDNA clones of NDV have been established internationally, they are limited to the Latasa strain and to the basic and clinical research level, which may be related to the technical capabilities of the vaccine production units in China.

Therefore, the technical problem to be solved by the present application is: how to develop infectious recombinant clonal viruses aiming at the II-type VII epidemic NDV strain DHN 3.

Disclosure of Invention

The invention aims to provide an infectious recombinant cloning method aiming at a II-type VII epidemic NDV strain DHN3, which can successfully clone recombinant viruses which are completely the same as original virus strains and have no mutation.

In order to achieve the purpose, the invention provides the technical scheme that: an infectious recombinant cloning method for a type II VII epidemic NDV strain DHN3, comprising the following steps:

step 1: constructing three types of helper plasmids, wherein target fragments on the helper plasmids are NP gene, P gene and L gene respectively;

step 2: constructing a DHN3 whole genome expression vector; the DHN3 whole genome obtained by artificial recombination is recombined into a vector plasmid to obtain a whole genome expression vector; the whole genome sequence of DHN3 is shown in a sequence table SEQ ID NO 1;

and step 3: co-transfecting the three auxiliary plasmids and a DHN3 whole genome expression vector to a BHK-21 cell to obtain virus liquid containing recombinant virus rDNN 3;

the NP gene is shown in a sequence table SEQ ID NO: the 1 middle position is 1-1591 nt; the P gene is shown in a sequence table SEQ ID NO: the position in the 1 is 1925 and 3109 nt; the L gene is shown in a sequence table SEQ ID NO: the 1 position is 8166 + 15192 nt.

In the above infectious recombinant cloning method against the II type VII epidemic NDV strain DHN3, the vector involved in the helper plasmid in the step 1 is pXJ40 series plasmid or pcDNA 3; the vector plasmid in the step 2 is a pBR322 series plasmid or a pACYC and pBAD series plasmid.

In the above infectious recombinant cloning method for the circulating NDV strain type II VII DHN3, the following steps are included:

step 1: constructing helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L, wherein target fragments of the helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L are NP gene, P gene and L gene respectively, and a vector is pXJ 40;

step 2: constructing a whole genome expression vector pBR322-DHN 3; the DHN3 whole genome obtained by artificial recombination is recombined into pBR322 plasmid to obtain a whole genome expression vector pBR322-DHN 3; the DHN3 whole genome is the whole genome of II-type VII-type newcastle disease virus;

and step 3: helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L and a whole genome expression vector pBR322-DHN3 are co-transfected into BHK-21 cells to obtain virus liquid containing recombinant virus rDNN 3.

In the above infectious recombinant cloning method for the type II VII epidemic NDV strain DHN3, the step 2 is specifically:

step 21: establishing a pBR322-Base vector; introducing a fragment capable of homologous recombination with the whole genome of DHN3 into a pBR322 plasmid; the fragment has homology arms corresponding to the 3 'end and the 5' end of the DHN3 whole genome;

step 22: constructing a transition vector; the transition vector is plasmid pBR322-PNP, plasmid pBR322-PDP and plasmid pBR322-LPD 3; the target fragment of the plasmid pBR322-PNP comprises NP, MINI and P genes; the target fragment of the plasmid pBR322-PDP comprises P, PD1 and PD2, PD3 genes; the target fragment of the plasmid pBR322-LPD3 comprises L1, L2, L3, L4 and PD3 genes;

step 23: constructing DHN3 whole genome DHN 3-A; carrying out enzyme digestion on the plasmid pBR322-PNP, the plasmid pBR322-PDP and the plasmid pBR322-LPD3 to obtain gene fragments PNP, LPD3 and PDP, and connecting the gene fragments PNP, LPD3 and PDP through T4 ligase to obtain DHN3 whole genome DHN 3-A;

step 24: constructing a plasmid fragment with a homology arm; carrying out PCR amplification by taking the pBR322-Base vector in the step 21 as a template to obtain a plasmid fragment with a homology arm;

step 25: constructing a whole genome expression vector pBR322-DHN 3; carrying out homologous recombination on the plasmid fragment with the homologous arm in the step 24 and the DHN3 whole genome DHN3-A in the step 23 to obtain a plasmid pBR322-DHN3 with the DHN3 whole genome DHN 3-A;

the MINI gene is shown in a sequence table SEQ ID NO: the 1 position is 1414-; the PD1 gene is shown in a sequence table SEQ ID NO: the 1-position is 2935-; the PD2 gene is shown in a sequence table SEQ ID NO: the 1 middle position is 4838 and 6454 nt; the PD3 gene is shown in a sequence table SEQ ID NO: the 1 position is 6261 and 8283 nt; the L1 gene is shown in a sequence table SEQ ID NO: the position in the 1 position is 8166 + 10709 nt; the L2 gene is shown in a sequence table SEQ ID NO: the 1-position is 10174 and 12299 nt; the L3 gene is shown in a sequence table SEQ ID NO: the 1-position is 12238-; the L4 gene is shown in a sequence table SEQ ID NO: position 14214- > 15192nt in 1.

In the above infectious recombinant cloning method for the type II VII epidemic NDV strain DHN3, the step 3 is specifically:

co-transfecting BHK-21 cells with plasmids capable of expressing T7RNA polymerase, helper plasmids pXJ40-NP, pXJ40-P, pXJ40-L and a whole genome expression vector pBR322-DHN 3; obtain virus liquid containing recombinant virus rDNN 3.

In the above infectious recombinant cloning method for the type II VII epidemic NDV strain DHN3, the step 21 is specifically:

introducing 1T 7 promoter, 1T 7 terminator and 1 HDV Ribozyme into pBR322 plasmid; partial base HC1 introduced to the 3' end of DHN3 downstream of the T7 promoter; the base HC2 in the 5' terminal part of DHN3 was introduced upstream of HDV Ribozyme; wherein, the position sequence of the base HC1 in the DHN3 whole genome sequence is 15192-15159 nt; the base HC2 has a position sequence of 141-1nt in the whole genome sequence of DHN 3.

In the above infectious recombinant cloning method for the type II VII epidemic NDV strain DHN3, the step 22 is specifically:

step 221: preparation of the pBR322 plasmid fragment: the pBR322 plasmid is subjected to double enzyme digestion by Hind3 and Nhe I, and then is recovered by gel for later use;

step 222: preparing a target fragment; the target fragment is NP, MINI, P, PD1, PD2, PD3, L1, L2, L3 and L4 genes;

step 223: the pBR322 plasmid fragment and the corresponding target fragment are linked by recombinase to obtain the corresponding plasmids pBR322-PNP, pBR322-PDP and pBR322-LPD 3.

In the above infectious recombinant cloning method for the circulating NDV strain type II VII DHN3, the step 23 is:

plasmid pBR322-PDP and plasmid pBR322-LPD3 are digested separately with BtgZ I to recover gene fragment LPD and PDP; the vector pBR322-PNP is subjected to double enzyme digestion by BtgZ I and Hind3 to recover a gene fragment PNP;

the gene fragment LPD, the gene fragment PDP and the gene fragment PNP are connected in vitro through T4 ligase to obtain DHN3 whole genome DHN 3-A.

The invention has the beneficial effects that:

the method of the invention can successfully clone the recombinant virus which is completely the same as the original virus strain and has no mutation.

The total genome of DHN3 is 15192nt, and if the full-length cDNA is obtained by RT-PCR at one time, the difficulty is that the general commercially available DNA polymerase can hardly synthesize DNA fragments larger than 10k, and the random mutation can be easily introduced in the PCR process. The DHN3 whole genome was therefore divided into 3 fragments, which were cloned separately into the pBR322 vector. The size of the fragment is determined based on the BtgZ I cleavage site it holds to facilitate subsequent in vitro DNA fragment ligation.

The invention adopts helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L and a whole genome expression vector pBR322-DHN3 to prepare virus liquid, and the main reasons are as follows: because the whole genome expression vector pBR322-DHN3 transfected into cells is limited, enough structural and functional proteins cannot be synthesized in cells in a short period of time; the components necessary for viral replication and assembly can be provided in a short period of time by co-transfecting helper genes.

Drawings

FIG. 1 is a diagram showing the alignment of the sequencing result of the first embodiment of the present invention with all published NDV sequences.

FIG. 2 is a micrograph showing that NDV-infected BHK-21 cells undergo significant cell fusion according to the first embodiment of the present invention;

FIG. 3 is a diagram showing the relationship between the L gene and sequencing plasmids pMD19-L1, pMD19-L2, pMD19-L3 and pMD19-L4 in the first embodiment of the present invention;

FIG. 4 is an electrophoretogram of the PCR product of helper plasmid pXJ40-L according to a first embodiment of the present invention;

FIG. 5 is an electrophoretogram of the PCR product of helper plasmid pXJ40-L according to a first embodiment of the present invention;

FIG. 6 is a restriction electrophoresis diagram of helper plasmid pXJ40-L according to the first embodiment of the present invention;

FIG. 7 is a plasmid schematic diagram of plasmid pXJ40-L, which is a first embodiment of the present invention;

FIG. 8 is a plasmid schematic diagram of plasmid pXJ40-P, which is a first embodiment of the present invention;

FIG. 9 is a plasmid schematic diagram of the plasmid pXJ40-NP according to the first embodiment of the present invention;

FIG. 10 is a plasmid schematic diagram of a pBR322-Base vector according to the first embodiment of the present invention;

FIG. 11 is a plasmid schematic diagram of plasmid pBR322-PNP according to the first embodiment of the present invention;

FIG. 12 is a plasmid diagram of plasmid pBR322-PDP according to the first embodiment of the present invention;

FIG. 13 is a plasmid schematic diagram of plasmid pBR322-LPD3 according to the first embodiment of the present invention;

FIG. 14 is a schematic structural diagram of a full-length DHN3-A according to a first embodiment of the present invention;

FIG. 15 is an electrophoretogram of positive colonies A1-A3 of the first example of the present invention;

FIG. 16 is a restriction enzyme electrophoresis diagram of the whole genome expression vector pBR322-DHN3 according to the first embodiment of the present invention;

FIG. 17 is a plasmid schematic diagram of the whole genome expression vector pBR322-DHN3 according to the first embodiment of the present invention;

FIG. 18 is a plasmid schematic diagram of the whole genome expression vector pBR322-DHN3 according to the first embodiment of the present invention;

FIG. 19 is a graph showing the growth characteristics of DHN3 virus, rDNN virus, and a blank control according to a first embodiment of the present invention;

FIG. 20 is a colony electrophoretogram of PCR of pXJ40DE3 plasmid in accordance with the first embodiment of the present invention;

FIG. 21 is a plasmid schematic diagram of a pBR322 plasmid fragment according to the first embodiment of the present invention;

FIG. 22 is a plasmid schematic of plasmid pXJ40 according to the first embodiment of the present invention;

FIG. 23 is a plasmid schematic of plasmid pXJ40-DE3, which is one embodiment of the present invention.

Detailed Description

The invention will be described with reference to specific embodiments: it is to be understood that these specific embodiments are merely illustrative of the invention and are not to be construed as limiting the invention. While those skilled in the art can fully appreciate the improvements to the embodiments and features of the present invention that can be made without departing from the scope of the present invention, such improvements and modifications are intended to be included within the scope of the present invention.

Example one

First, main instrument and reagent

The THZ-100 type electric heating constant temperature incubator is purchased from Shanghai-Hengchun scientific instruments Co.

Model a17105653 clean bench was purchased from santai air technologies, suzhou.

The DK-8D type three-hole electric heating constant temperature water tank is purchased from Shanghai Hengscientific instruments Co.

The THZ-100 type constant temperature incubator was purchased from Shanghai-Hengchang scientific instruments, Inc.

A hail refrigerator model BCD-579WE was purchased from hail.

5424R type centrifuge; an Eppendorf PCR instrument; thermo1300 SERIES A2 biosafety cabinets, all purchased from Eppendorf.

DNA/RNA Co-extraction Kit (AP-MN-BF-VNA-250G) was purchased from AXYGEN, and TIAN prep Mini plasma Kit (DP103-03) was purchased from Tiangen, Inc. Gel Extraction Kit (D2500-02) was purchased from OMEGA; M-MLVRT (2641A) was purchased from TAKARA; RRIs (2313A) were purchased from TAKARA; random 6primer (3801) was purchased from TAKARA; agarose (E0301) was purchased from TSINGK; 0.25% Trypsin-EDTA (25200-056), DMEM basic (C11995500BT) from Gibco; lipofectamine LTX and Plus Reagent (15338-100) from Invitrogen; FBS (10099-141C) was purchased from Gibco; Premix-Taq (RR902A) was purchased from TAKARA; pen streppellicin Streptomyces (15140-122) was purchased from Gibco; primer star GXL (R050) was purchased from TAKARA; BtgZ I (R0703S), T4 DNA Ligase (M0202M) from NEB; other restriction enzymes, ligase, were purchased from TAKARA. Clon express Multi One Step Cloning Kit (C113), Novomedium; BL21(DE) was purchased from Merck; the vector pMD19 was purchased from Takara and pBR322 was purchased from NEB.

Second, virus separation, purification, sequencing and analysis classification

(a) In order to develop a recombinant vaccine aiming at the currently popular NDV, a disease material NDV (QTCF1) is collected from a chicken farm, a proper amount of normal saline is added into the disease material, the disease material is fully ground, the centrifugation is carried out at 12000rpm for 10 minutes, then the supernatant is taken and filtered through a 0.4 mu m filter, 1 percent of SP double antibody is added into the filtrate, the filtrate is directly inoculated into the allantoic cavity of a chick embryo membrane without dilution, and the addition amount is 100 mu l/piece;

then inoculating and breeding in chorioallantoic cavity of SPF chick embryo, and collecting allantoic fluid after chick embryo dies.

(b) The virus contained in this allantoic fluid was plaque-purified from BHK-21 cells, and the purified virus was designated DHN 3. The specific operation is as follows:

double distilled water is used to prepare 3% agarose solution, and the solution is placed at 4 ℃ for standby after high pressure. Before use, the mixture is heated for 2-3 minutes by a microwave oven to melt, and prepared into agarose liquid by 3% agarose 2ml and 2% FBS DMEM 24ml, and the agarose liquid is put into dry bath at 40 ℃ for standby.

2% FBS + DMEM + 1% SP antibiotic culture solution is prepared for standby.

DHN3 virus recovered from allantoic fluid was cultured in DMEM at a 1: diluting 10 to 6 gradients to obtain infection liquid 10^-1，10^-2，10^-3，10^-4，10^-5，10^-6And (5) standby.

BHK-21 cells were cultured in 6-well dishes, and 200. mu.l of the infection solution was added after the cells were 90% full, and 800. mu.l of DMEM was supplemented to make the final volume of each well 1 ml. After gentle shaking, the mixture was incubated at 37 ℃ for 2 hours. The incubation was discarded and washed 2 more times with PBS. Plaques were observed after 2.5ml of agarose solution was added to each well for 4-6 days.

Selecting 1 plaque with moderate size to respectively infect BHK-21 cells, and extracting total RNA of the cells when cytopathic effect is obvious (NDV infected BHK-21 cells can generate obvious cell fusion phenomenon (as shown in figure 2).

This total RNA was used as a template, and reverse transcription was performed using a non-specific primer (Random 6primer (3801)) to obtain a first cDNA.

Then using the cDNA as a template and using specific primers and ExTaq enzyme for PCR (wherein the specific primers are shown in the following attached table 1 for corresponding primers of each plasmid, and the reaction conditions of PCR are shown in tables 2, 3 and 4);

these PCR products were purified and cloned into pMD19 vector, and then sequenced with primers (M13-F, M13-R) at both ends of pMD19 vector, respectively. A total of 10 plasmids covered the whole genome of DHN3, detailed in attached Table 1.

TABLE 1 plasmid Table for detection of DHN3 sequences

Primer L1-F is shown in SEQ ID NO: 2; primer L1-R is shown in SEQ ID NO: 3; primer L2-F is shown in SEQ ID NO: 4; primer L2-R is shown in SEQ ID NO: 5; primer L3-F is shown in SEQ ID NO: 6; primer L3-R is shown in SEQ ID NO: 7; primer L4-F is shown in SEQ ID NO: 8; primer L4-R is shown in SEQ ID NO: 11; primer PD1-F is shown in SEQ ID NO: 9; primer PD1-R is shown in SEQ ID NO: 10; primer PD2-F is shown in SEQ ID NO: 12; primer PD2-R is shown in SEQ ID NO: 13; primer PD3-F is shown in SEQ ID NO: 14; primer PD3-R is shown in SEQ ID NO: 15; primer MINI-F is shown in SEQ ID NO: 16; the primer mini-r is shown in SEQ ID NO: 17; primers P-F are shown in SEQ ID NO: 18; the primer P-R is shown in SEQ ID NO: 19; primer NDV-ST-W R is shown in SEQ ID NO: 20; the primer NP-LB-R is shown in SEQ ID NO: 21; primer M13-F is shown in SEQ ID NO: 22; primer M13-R is shown in SEQ ID NO: 23.

TABLE 2 reaction conditions for PCR

TABLE 3 amplification primers in PCR reaction conditions

TABLE 4 application formulation table of different enzymes in reaction conditions of PCR

Remarking:

r050PCR reaction procedure

RR902 PCR reaction procedure

3. The preparation of the PCR reaction system and the setting of the reaction procedure according to the present invention were carried out in the same manner as described above.

Only the annealing temperature and extension time during PCR will be adjusted for each specific primer and fragment (as described in the above table), and nothing else will be changed. The process of the PCR reaction will not be described in detail below.

The sequencing results were aligned to all published NDV sequences by NCBI Blast software, and the results are shown in figure 1. The conclusion is that the DHN3 Newcastle disease virus genome is 15192nt in full length and belongs to type II VII NDV.

To ensure the sequence is accurate, the above experiment was repeated 3 times from virus infection to sequencing results analysis. The sequencing results were identical, confirming that a purified strain of DHN3 was obtained from this plaque.

The DHN3 sequence is shown in a sequence table SEQ ID NO: 1.

thirdly, constructing recombinant virus rDNN 3

3.1 construction of plasmids required for obtaining recombinant viruses

3.1.1 construction of helper plasmids

The NP, P, and L genes were cloned into pXJ40 vectors to obtain helper plasmids pXJ40-NP, pXJ40-P, and pXJ40-L, respectively. The pXJ40 plasmid contains the CMV promoter and SV40 polyadenylation A signal fragment, so that foreign genes can be efficiently transcribed by cellular DNA polymerase II and then translated into NP, P, and L proteins, which are proteins used to assemble active virions.

The specific method is as follows:

construction of helper plasmid pXJ40-L

The foregoing sequencing plasmids pMD19-L1, pMD19-L2, pMD19-L3 and pMD19-L4 covered the entire L gene of DHN3, as shown in FIG. 3 below.

By designing 4 specific primers for band homologous recombination sequences (see attached table 5 in particular), and respectively taking pMD19-L1, pMD19-L2, pMD19-L3 and pMD19-L4 as templates, fragments L1, L2, L3 and L4 which can be used for cloning L complete sequences into pXJ40 vectors are amplified. The 5 'end of L1 has a homology arm with pXJ40 BamH1 terminal, and the 3' end of L4 has a homology arm with pXJ40 Pst1 terminal.

The vector pXJ40 was double digested with BamH1 and Pst1 and recovered by gel purification.

The 4 fragments were ligated with vector pXJ40 double-digested with BamH1/Pst1, and homologous recombination was performed with recombinase (Clon express Multi One Step Cloning Kit (C113)) to obtain a recombinant product.

The recombinant product was transformed into DH5 alpha cells and screened by single colony PCR. The correct PCR product was amplified to 2110bp with primer PXJ40-F (pXJ40-F: GCAACGTGCTGGTTATTGTG)/P-L1-R (P-L1-R: GGACAGTTGACTCATTGCTAACATA); the correct PCR product amplified with the primer P-L3-F/P-L3-R2027 bp. is demonstrated in FIGS. 4 and 5: 8 colonies are detected, and PCR products with correct molecular weight are generated by 8 positive bacteria;

primer PXJ40-F is shown in SEQ ID NO: 24; primer P-L1-R is shown in SEQ ID NO: 25;

further culturing and amplifying one of the strains, and extracting plasmid DNA. Then the enzyme is cut by BamH1/Pst1, the correct cut products should be 6230bp and 5081 bp. The results of the experiment are shown in FIG. 6 below, and it was confirmed that the plasmid contained a DNA fragment of the correct molecular weight, and the schematic diagram of the plasmid pXJ40-L is shown in FIG. 7.

TABLE 5 PCR primers used to create pXJ40-L

Primer P-L1-BamH1-F is shown in SEQ ID NO: 26; primer P-L2-F is shown in SEQ ID NO: 27; primer P-L2-R is shown in SEQ ID NO: 28; primer P-L3-F is shown in SEQ ID NO: 29; primer P-L3-R is shown in SEQ ID NO: 30, of a nitrogen-containing gas; primer P-L4-F is shown in SEQ ID NO: 31; primer P-L4-Pst1-R is shown in SEQ ID NO: 32.

TABLE 6 construction of the formulation Table for plasmid pXJ40-L

Remarking:

1. for the other two helper plasmids, pXJ40-NP, pXJ40-P was constructed by using a method of ordinary ligase ligation transformation, and not by using a method of homologous recombination.

Construction of plasmid pXJ40-NP, pXJ40-P

2.1 the pXJ40 plasmid was digested simultaneously with EcoRI and XhoI, and the excised fragment (4288bp) was used as a plasmid fragment for the construction of pXJ40-NP, pXJ 40-P.

2.2 amplification of NP fragments (R050,60 ℃ annealing temperature, 1min30s extension time) using NP-PXJ-0F2/NP-PXJ-1591R2 (see Table 9). P gene (R050, annealed at 60 ℃ C., extended 1min15s) was amplified using P-PXJ-1083F2/P-PXJ-3080R 2. The primers P-PXJ-0F2 and P-PXJ-1083F2 in the two pairs of primers for amplifying NP and P genes contain the enzyme digestion sequence of EcoRI; the primer NP-PXJ-1591F2 and the primer P-PXJ-3080R2 contain Xho I sequences. Thus, using these two pairs of primers to amplify the NP and P genes, respectively, the amplified products contain EcoRI and XhoI cleavage sites at both ends, respectively. The product amplified using these two pairs of primers was then double digested with EcoRI and Xho I, so that the cleaved product contained cohesive ends of EcoRI and Xho I.

2.3 the enzyme cutting products of the linearized pXJ40 fragment containing the cohesive ends of EcoRI and Xho I and the NP and P fragments containing EcoRI and Xho I respectively are connected, transformed and checked by selecting bacteria. Thus, plasmid pXJ40-NP, pXJ40-P was constructed.

3. When pXJ40-L plasmid is constructed, R050 high fidelity enzyme is used for amplifying each fragment, the annealing temperature is 60 ℃, and the extension time calculation method and the amplification system are calculated according to the R050 enzyme.

4. For the identification of plasmid pXJ40-L, primers pXJ40-F and P-L1-R were used, and RR902 enzyme was used. Preparation of reaction liquid the reaction procedure and the extension time were calculated as described above, and the annealing temperature of the pair of primers was 57 ℃.

The following Table 7 shows the digestion of plasmid pXJ40 and the digestion of the amplified products of NP and P genes. In addition, the sequence 8 is the ligation process of the digestion product of NP and P genes and the digestion product of pXJ40 plasmid. It should be noted that PCR products and enzyme-cleaved products of NP and P genes and enzyme-cleaved products of pXJ40 plasmid were recovered by agarose gel electrophoresis and then subjected to the next step.

TABLE 7 restriction enzyme digestion Process of plasmid pXJ40 and restriction enzyme digestion Process of amplified products of NP, P genes

TABLE 8 parameter Table of ligation of the cleavage products of NP and P genes with the cleavage product of pXJ40 plasmid

The P gene is inserted between EcoR1 and Xho1 of pXJ 40; the NP gene was inserted between EcoR1 and Xho1 of pXJ 40. See plasmid schemes 8 and 9.

TABLE 9 primers used to establish pXJ40-P, pXJ40-NP

Primer P-PXJ-1893F2 is shown in SEQ ID NO: 33; primer P-PXJ-3080R2 is shown in SEQ ID NO: 34; primer NP-PXJ-0F2 is shown in SEQ ID NO: 35; primer NP-PXJ-1591R2 is shown in SEQ ID NO: 36;

the vector plasmids pXJ40, pXJ40 mentioned above and in many places below are very widely used vector plasmids with abundant restriction sites, which are used pXJ40 in the construction of a large number of plasmids.

The vector plasmid pXJ40 used in the invention is derived from the national institute of agriculture university of south China Liudingxiang professor laboratory of group microorganisms, the plasmid schematic diagram is shown in figure 22, pXJ40-NEW shown in figure 22 is pXJ40, and the sequence of the plasmid is shown in a sequence table SEQ ID NO: 66; the method can be obtained by artificial sequence synthesis according to the sequence table by a person of ordinary skill in the art. The group microorganisms of university of south China university center Liudingxiang professor's laboratory may provide the vector plasmid of the plasmid structure diagram as shown in FIG. 22.

Meanwhile, the vector plasmid pXJ40 can also be obtained by converting other commercialized plasmids in the field, and specifically, pXJ40 used in the embodiment can be prepared by using a commercial plasmid pXJ40-flag, and the transformation principle is as follows: to avoid the effect of Flag on viral protein function; we obtained pXJ40-New by engineering the commercial plasmid pXJ40-Flag to remove Flag.

pXJ40-flag has at least the following two sources;

channel one and laboratory preservation; pXJ40-flag is preserved in Liu Ding Xiang professor laboratory of the group microorganism center of the university of agriculture in south China, and pXJ40-flag plasmid can be provided in Liu Ding Xiang professor laboratory of the group microorganism center of the university of agriculture in south China; pXJ40-Flag is described in the published literature of professor Liu's auspicious, specifically "pXJ 40F (with a Flag tag)", which is published as: journal of virology, Dec.2009, p.12462-12472, Inhibition of Protein Kinase R Activation and alignment of GADD34 Expression Play a Synergistic Role in purifying Coronavir reproduction by Maintaining De Novo Protein Synthesis in viruses-induced Cells author Liang, Weak, Liu Ding Xiang.

Channel two, purchased from prohibitin biotechnology (Beijing) Inc., under the reference pXJ 40-flag.

The preparation method comprises the following specific steps:

step 1: preparation of plasmid fragment: 2ug pXJ40Flag +1ul EcoR1+1ul Bsa1+5ul buffer added water to 50ul and incubated at 37 ℃ for 2 hours; recovering 2998bp fragment from the gel for later use;

step 2: PCR amplification of Flag-free fragment: a1322 bp fragment (annealing temperature 60 ℃ C., extension time 1min30 sec) was amplified by PCR using 2ng of pXJ40Flag as a template and a primer dFlagF: ACTATAGGGCGAATTCGGATCCAAGCTTCTCG/Bsa1R: TGAGCGTGGGTCTCGCGGT and high-fidelity DNA polymerase (TAKARA R050), and the gel was recovered for use.

And step 3: using Clon express multiple One Step Cloning Kit (Novozan C113), 2998bp fragment 50ng obtained in the Step 1, 1322bp fragment 22ng obtained in the Step 2, buffer 4ul and enzyme 2ul, and adding water to 20 ul; incubate at 37 ℃ for 30 minutes.

And 4, step 4: the recombinant product was transformed into DH 5. alpha. (conventional method, TAKARA), and screened for ampicillin culture. The plasmid was prepared conventionally and verified by sequencing with primer pXJ40R: AGCGGAAGAGTCTAGAGTCG. The plasmid containing the correct sequence (no Flag sequence) was pXJ 40-New.

3.1.2 construction of pBR322-Base vector

To obtain recombinant viruses, 1 basic plasmid (pBR322-Base) was first constructed to obtain single-stranded minus-strand RNA from the whole genome.

In order to obtain higher transcription level and ensure that the transcribed RNA does not contain any exogenous ribonucleic acid, 1T 7 promoter, 1T 7 terminator and 1 HDV Ribozyme are respectively introduced into a pBR322 plasmid, so that DHN3 whole genome minus strand full-length RNA can be accurately synthesized under the action of exogenous T7RNA polymerase.

In order to facilitate the integration of the whole genome of DHN3 into the pBR322 vector by homologous recombination, HC1 at the 3' end of DHN3 was introduced downstream of the T7 promoter; the base HC2 in the 5' terminal part of DHN3 was introduced upstream of HDV Ribozyme; the position sequence of the base HC1 in the DHN3 whole genome sequence is 15192-15159 nt; the base HC2 has a position sequence of 141-1nt in the whole genome sequence of DHN 3. (see plasmid scheme 10)

DNA fragments containing T7 terminator, HDVRibozyme, HC2, HC1 and T7 promoters are artificially synthesized and combined onto a pBR322 vector in a homologous recombination mode to obtain pBR322-T7Pro-HDV _ Ter, namely a basic plasmid pBR322-Base (a plasmid schematic diagram 10).

3.1.3 establishment of 3 subgenomic transition vectors

The total genome of DHN3 is 15192nt, and if the full-length cDNA is obtained by RT-PCR in one time, the whole genome is difficult to obtain (generally, the commercially available DNA polymerase has difficulty in synthesizing DNA fragments larger than 10 k), and random mutation is easy to introduce in the PCR process. The DHN3 whole genome was therefore divided into 3 fragments, which were cloned separately into the pBR322 vector. The size of the fragment is determined based on the BtgZ I cleavage site it holds to facilitate subsequent in vitro DNA fragment ligation.

The specific construction method comprises the following steps:

A. preparation of plasmid fragment: the pBR322 vector (see FIG. 21) was digested simultaneously with Hind3 and Nhe I, and recovered by gel.

B. Preparing a target gene fragment: the primers with the homologous arms and the corresponding templates (see the following table 10) are respectively used for amplifying DNA fragments with the homologous arms under the action of high-fidelity DNA polymerase, and the DNA fragments are recovered for standby after being verified by running DNA gel.

C. The target gene fragment and the pBR322 plasmid fragment are linked by a recombinase. After bacterial transformation, single colony PCR initial selection, plasmid DNA amplification, plasmid DNA restriction enzyme digestion, DNA gel check, and final sequencing verification.

The fragments of interest for constructing the pBR322-PNP plasmid include NP, MINI, and P.

The target fragments for constructing the pBR322-PDP plasmid include P, PD1 and PD2, PD 3.

The fragments of interest for the construction of the pBR322-LPD3 plasmid include L1, L2, L3, L4 and PD 3.

The above 3 intermediate plasmids are shown in FIGS. 11-13, respectively, wherein FIG. 11 is: pBR322-PNP plasmid diagram; FIG. 12 is a diagram: schematic representation of pBR322-PDP plasmid; FIG. 13 is a schematic diagram of pBR322-LPD3 plasmid.

TABLE 10 template and primer tables related to the target fragments

The primer C-Sac11BtNhe-ST-R is shown in SEQ ID NO: 37; the primer C-NP-F is shown in SEQ ID NO: 38; the primer C-Mini-R is shown in SEQ ID NO: 39; primer C-Mini-F is shown in SEQ ID NO: 40; the primer C-P-R is shown in SEQ ID NO: 41; primer C-HindBt-P-F is shown in SEQ ID NO: 42; the primer C-BtNhe-P-R is shown in SEQ ID NO: 43; primers C-P-F are shown in SEQ ID NO: 44; primer C-PD1-R is shown in SEQ ID NO: 45, a first step of; primer C-PD1-F is shown in SEQ ID NO: 46; primer C-PD2-R is shown in SEQ ID NO: 47; primer C-PD2-F is shown in SEQ ID NO: 48; primer C-PD3-R is shown in SEQ ID NO: 49; primer C-HindBtg-PD3-F is shown in SEQ ID NO: 50; primer C-BtNhe-PD3-R is shown in SEQ ID NO: 51; primer C-PD3-F is shown in SEQ ID NO: 52; primer C-L1-R is shown in SEQ ID NO: 53; primer P-L2-R is shown in SEQ ID NO: 54, a first electrode; primer P-L3-F is shown in SEQ ID NO: 55; primer C-HindBtNot-L4-F is shown in SEQ ID NO: 56;

TABLE 11 construction of plasmids

Remarking:

1. after the target fragments required for constructing the PBR322-PNP plasmid, the PBR322-PDP plasmid and the PBR322-LPD3 plasmid were amplified respectively, the plasmids were mixed according to the recombination system in Table 11 above, and then the corresponding plasmids were constructed by performing homologous recombination.

2. According to the construction strategy of the PBR322-DHN3 plasmid, the viral genome contains two BtgZ I enzyme cutting sites, and the whole viral genome is divided into 3 large fragments according to the two enzyme cutting sites, namely, three large fragments contained in three plasmids of PBR322-PNP, PBR322-PDP and PBR322-LPD3 are constructed. Meanwhile, when basic sequencing is carried out on a virus genome, the whole virus genome is divided into 10 small fragments (NP, P, PD1, PD2, PD3, L1, L2, L3 and L4), and BtgZ I enzyme cutting sites are just positioned on the P fragment and the PD3 fragment, so that P fragments and PD3 are named as P left, P right, PD3 left and PD3 right when plasmids of PBR322-PNP, PBR322-PDP and PBR322-LPD3 are amplified.

3. Since the L protein is the nucleocapsid protein of NDV virus, after the completion of plasmids PMD19-L1, PMD19-L2, PMD19-L3 and PMD19-L4, plasmid PXJ40-L is first constructed and used as an accessory protein for rescuing viruses. Thus, PXJ40-L was one of the templates used in the construction of the PBR322-LPD3 plasmid, which already contains the entire sequence of the L gene.

Dividing the L gene into 2 segments for amplification in the process of constructing PBR322-LPD3, namely L1-2 and L3-4, (wherein, L1-2 refers to the continuous sequence of small fragments L1 and L2, which is constructed by a primer C-L1-R, P-L2-R and a template pXJ40-L, and L3-4 refers to the continuous sequence of small fragments L3 and L4, which is constructed by a primer P-L3-F, C-dBHintNot-L4-F and a template pXJ 40-L); and it should be pointed out that the primer C-HindBtNot-L4-F for amplifying L3-4 contains a homologous region required for constructing the PBR322-DHN3 plasmid and a BtgZ I enzyme cutting sequence, so that the virus genome sequence in the PBR322-LPD3 can be completely cut off by the BtgZ I, and the cut-off fragment is directly used for constructing the PBR322-DHN3 plasmid.

4. The PCR amplification reactions involved in the construction of the PBR322-PNP plasmid, PBR322-PDP plasmid, PBR322-LPD3 plasmid in Table 11 above all used enzymes PrimeSTARGXLDANMERase (R050, TAKARA), the reaction system referred to previously. According to the characteristics of the enzyme R050, in the amplification reaction, the pre-denaturation temperature and time are 95 ℃ and 3min, the denaturation temperature and time are 98 ℃ and 10s, the annealing temperature and time are 60 ℃ and 15s, and the extension temperature and time are 68 ℃ and the extension time is properly selected according to the proportion of 1000bp extension 60s according to the fragment size. The reaction system was 30. mu.l each.

5. In the experiments using the homologous recombination method, the kits used in the present invention are all Clon ExpressCardiosONTepConningkit (C113, Novozan), and will not be described again.

3.1.4 construction of the Whole genome expression vector pBR322-DHN3

Preparation of DHN3-a fragment: the fragment PNP, the fragment PDP and the fragment LPD3 are recovered from pBR322-PNP plasmid, pBR322-PDP plasmid and pBR322-LPD3 plasmid by enzyme digestion; and the fragment PNP, the fragment PDP and the fragment LPD3 are connected to obtain full-length DHN3-A for standby.

Specifically, referring to Table 12, pBR322-PNP plasmid was digested with BtgZ I and Hind3 to recover the fragment PNP;

the method for recovering fragment PDP from pBR322-PDP plasmid is as follows: the pBR322-PDP plasmid is subjected to single digestion by BtgZ I, and then the fragment PDP is recovered;

the method for recovering fragment LPD3 from pBR322-LPD3 plasmid includes the following steps: the pBR322-LPD3 plasmid is digested with BtgZ I to recover LPD 3;

TABLE 12 recovery formulation and Process parameters Table for each plasmid fragment

Remarking:

1. the preparation of the fragment PNP, the fragment PDP and the fragment LPD3 is carried out according to the enzyme digestion system and the time in the table; after the enzyme digestion is finished, a small amount of the enzyme is used for gel electrophoresis to observe whether the enzyme digestion is complete, if the enzyme digestion is incomplete, a little more enzyme is added properly or the incubation time is prolonged, or both the enzyme digestion and the incubation time are both required;

2. preparation of the fragment PNP requires cleavage by both Hind3 and BtgZ I enzymes. It should be noted that the two enzymatic reactions are performed separately, not simultaneously. The enzyme is firstly cut by Hind3, and the BtgZ I enzyme is used for enzyme cutting after the target product is recovered by agarose electrophoresis gel. Thus pBR 322-PNP' in the above table refers to the product of Hind 3.

3. A0.6% agar (TsingKe, TSJ001) gel containing 0.03% of the whole gold staining solution was prepared. 0.6 microgram of agar is added with 100 milliliters of TAE buffer solution and placed in a microwave oven to be heated for 1 to 2 minutes until the agar is completely melted, and after the agar is cooled to about 50 ℃, the whole gold staining solution (diluted by 1: 3000 times) is added and mixed evenly, and then glue is spread. Note that the stain adds a link that may inhibit the DNA fragments. All the above digestion products were separated by gel electrophoresis as described above. The gel was placed on a clean glass plate and the correct target fragment was cut off and recovered with a disposable stainless steel blade. Purification was performed using GelExtractionkit (D2500-02, OMEGA) according to the protocol provided in the kit.

The fragment PNP, the fragment PDP and the fragment LPD3 are connected in vitro by a high-concentration T4 ligase to obtain full-length DHN 3-A. The specific parameters refer to table 13. The plasmid structure of full-length DHN3-A is shown in FIG. 14;

TABLE 13 ligation formulation Table of pBR322-DHN3 plasmid

Remarking:

1. the fragment PNP, fragment LPD3 were used in the above table calculations and ligated.

2. The ligation product was directly purified using a kit (MiniBESTDNAfragmentpurifcation kit version4.0, TAKARA, 9761), and the purified product was run on an agarose electrophoresis gel to see if the ligation was complete.

Preparation of a plasmid fragment with homology arms: using the above pBR322-Base plasmid as a template, a special primer (A2-F: ATCGGATAG) containing a Base pairing with a homologous arm was usedAAGGTTCCCTCAGGTTC, respectively; A2-R: GGTCCTATAGTGAGTCGTATTAATG), PCR amplification was performed using the materials and according to the methods provided in the kit with Primer star GXL kit. Primer A2-F is shown in SEQ ID NO: 57; primer A2-R is shown in SEQ ID NO: 57;

the PCR amplification product of this step was purified by the above purification kit (Mini BESTDNA fragment purification kit version4.0, TAKARA, 9761) by the method provided in the kit to prepare a plasmid fragment having a homology arm.

The plasmid fragment and DHN3-A are subjected to homologous recombination to obtain a whole genome expression vector pBR322-DHN 3.

Specifically, it was performed by using a Kit purchased from Novovophilia recombination Kit (Clonexpressions One Step Cloning Kit, C113). The plasmid fragment with the homology arm and the DHN3 whole genome DNA fragment (i.e., the in vitro ligation product, DHN3-a) are taken to perform homologous recombination according to the method provided by the novispan recombination kit, and the specific formula parameters can refer to table 14.

TABLE 14 formulation Table for homologous recombination of plasmid fragments with DHN3-A

Reagent	Dosage of
		DHN3-A	200ng
pBR322-Base	91.02ng
		5×CE MultiS Buffer	4μl
Exnase MultiS	2μl
		ddH2O	up to 20μl

The recombinant product was transformed into DH5a competent cells. Using specific primers C-pBR322-R (C-pBR 322-R: GAAATTGCATCAACGCATATAGCGC); single colony PCR was performed with C-NP-F (C-NP-F: CATCTGGTTGCCCTTGCGGCTTGTTC) to obtain 3 positive colonies A1-A3, as shown in FIG. 15. Primer C-pBR322-R is shown in SEQ ID NO: 59; the primer C-NP-F is shown in SEQ ID NO: 60.

colony A1 was cultured for amplification and plasmid DNA was extracted. The A1 plasmid was digested with Sac1 for preliminary identification, and the correct digestion products should be 1 fragment of 8482bp, 1 fragment of 7065bp, 1 fragment of 3132bp, 1 fragment of 908bp, and 1 fragment of 32 bp. The results are shown in FIG. 16, which shows that the cleavage products agree with the results of the sequence estimation. The 32bp fragment was too small to be visualized in an agar electrophoresis gel. This plasmid was named pBR322-DHN 3. The plasmid was re-sequenced and amplified for future use after verification.

Plasmid structure of plasmid pBR322-DHN3 is shown in FIG. 17; plasmid structure of plasmid pBR322-DHN3 is shown in FIG. 18.

3.1.5 rescue the virus.

BHK-21 cells were plated on 30mm dishes overnight to 80-90% confluency. Was co-transfected with pXJ40DE3(DE3 expressing T7RNA polymerase) and pBR322-DHN3, pXJ40-NP, pXJ40-P and pXJ 40-L. After 4 days, the culture dish is placed in a-20 refrigerator for freeze thawing for 2 times, then the cells and the culture solution are collected into a sterile centrifuge tube, the centrifuge tube is centrifuged at 12000rpm for 10 minutes, and the supernatant is collected. Then 100. mu.l of the supernatant (containing rDNN 3) was further infected with BHK-21 cells, and cell fusion lesions were observed 2 to 3 days later. After further culturing for 1 day, the plates were frozen and thawed in a-20 freezer 2 times, and then collected and centrifuged at 12000rpm for 10 minutes, and the supernatant was collected. In order to accelerate virus amplification, virus rDNN 3P1 can be inoculated into SFP chick embryos, allantoic fluid is collected to obtain virus rDNN 3.

The transfection step involves the following amounts of the respective substances of the transfection solution:

the transfection liquid comprises a liquid A and a liquid B

Transfection was performed as provided by Lipofectamine LTX and Plus Reagent (15338-100), Invitrogen.

HN is on the surface of the Newcastle disease virus envelope and can adsorb red blood cells to cause agglutination of part of animal red blood cells. When the concentration of newcastle disease virus is high, it may further cause lysis of erythrocytes. According to the characteristics of the Newcastle disease virus, whether the Newcastle disease virus exists in the solution to be detected and the content of the virus can be detected through the experiment. The first generation rDNN 3 rescued by the method is used for collecting allantoic fluid as a sample to be detected after SFP chick embryo amplification, and the following erythrocyte coagulation experiment proves that the rDNN 3 recombinant virus has the same blood coagulation function as a mother strain. Both of them had the highest coagulation titer of 2⁸。

The specific test process is as follows:

1. using normal saline to treat a sample to be detected according to the ratio of 1: 10, performing system dilution;

2. adding 25 mul of physiological saline into each well of a 96-well plate;

3. adding virus solutions with different dilutions into corresponding wells, 25 μ l/well (one virus for each row, no virus solution added in the last row for comparison);

4. add 25. mu.l of 1% chicken red blood cells to each well;

blood coagulation was observed after 5.15-20 minutes.

The plasmid pXJDE3 referred to above is specifically as follows:

pXJ40DE3 plasmid is a T7RNA polymerase (DE3) expression plasmid. The DE3 gene was obtained from E.coli BL21(DE3) (Merck) by using the primers BamH1-DE3-F/BamH1-DE 3-R. Inserting the plasmid into a BamH1 site of a plasmid pXJ40 through homologous recombination; pXJ40DE3 plasmid structure diagram is shown in FIG. 23.

pXJ40DE3 plasmid preparation steps

Step 1: extraction of E.coli BL21(DE3) DNA: conventional bacterial DNA extraction methods.

Step 2: preparation of the DE3 fragment by PCR: using bacterial DNA of escherichia coli BL21(DE3) as a template, and performing amplification by using high fidelity DNA polymerase (GXL) through primers:

BamH1-DE3-F：actcactatagggcgaattcggatccgccatgaacacgattaacatcgc；

BamH1-DE 3-R: taagatctggtaccgagctcctgcagttacgcgaacgcgaagtccgactc amplification is carried out.

Primer BamH1-DE3-F is shown in SEQ ID NO: 61;

primer BamH1-DE3-R shown in SEQ ID NO: 65;

GXL enzyme (R050), annealing temperature 60 ℃ and extension time 2 min 36 sec. The product is purified for later use after gel electrophoresis verification.

And step 3: preparing a carrier: pXJ40 is cut by PstI and BamHI enzyme and recovered by glue for standby, and the specific formula and process refer to Table 15.

TABLE 15 double enzyme digestion formulation process table of vector pXJ40

pXJ40	1μg
		Quick Cut PstⅠ	1μl
Quick Cut BamHⅠ	1μl
		10×QuickCut Buffer	2μl
ddH20	up to 20μl
		Temperature of	37℃
Time	3h

And 4, step 4: the DE3 fragment was recombined into pXJ40 vector, the specific process and formulation are shown in table 16 below.

TABLE 16 Process formulation Table for recombination of DE3 fragment into pXJ40 vector

And 5: the recombinant product was transformed into DH 5. alpha. by plating with 50ug/ul ampicillin. The following day 8 colonies were picked from the plate for PCR validation.

The verification primer is pXJ40-F: GCAACGTGCTGGTTATTGTG; DE3-R: GAAGTCCGACTCTAAGATGTCACG, ExTaq enzyme (RR902) was used, annealing temperature 57 ℃, extension 2' 42 "(target fragment size 2723bp) results are shown in FIG. 20:8 bacteria have 3 strong positives (L1, L5, L7)1 weak positives (L4). The plasmid was extracted after the L1 amplification culture, and the sequencing was verified without error. Primer DE3-R is shown in SEQ ID NO: 62, a first step of mixing;

fourth, identify rDNN 3

4.1 Gene stability

The virus rDNN 3 rescued above is used to infect BHK-21 cells after 3 passages by chick embryo, and total RNA of the cells is extracted when cytopathic effect is obvious.

The first cDNA is obtained by reverse transcription using a specific primer, and PCR is carried out using the cDNA as a template and a specific primer (JF-F: CCTTGCAGCTGCAGGAATTG; JF-R: GCTCTATACAGTATGAGGTGTCAAG) and DNA polymerase. The PCR product was purified and then sequenced using primers (JF-F: CCTTGCAGCTGCAGGAATTG). The results showed no difference from the original strains. Indicating that the DHN 3F gene was stable during viral rescue and no natural mutation occurred. Primer JF-F is shown in SEQ ID NO: 63; primer JF-R is shown in SEQ ID NO: 64.

4.2 fusion lesion status of BHK-21 cells infected by recombinant virus rDNN 3

Referring to FIG. 19, there are three columns A, B, C, in which column A represents the morphology of DHN3 virus 12h after infecting BHK-21 cells; the B column is the cell shape after the BHK-21 cells are infected by rDNN 3 for 12 hours; the C column is the form 12h after BHK-21 cells are not infected with the virus.

The arrows in the figure indicate the fusion lesions that appear when BHK-21 cells are infected with DHN3 or rDHHN 3

DHN3 refers to virus that was isolated, blinded and plaque-purified on BHK-21 cells; the virus was involved in both the sequencing and analytical classification steps.

rDNN 3 refers to a recombinant virus, i.e., a virus rescued by recombinant cloning herein.

As can be seen in FIG. 20, both the recombinant virus rDHHN 3 and its parent virus DHN3 were able to infect BHK-21 to form a cell fusion.

In addition, the growth characteristics and the toxicity of rDNN 3 and DHN3 are compared, and no obvious difference is found between the rDNN 3 and the DHN 3.

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Sequence listing

<110> Chongqing research institute of BioIndustrial technology, Inc

<120> infectious recombinant cloning method aiming at II-type VII-type epidemic NDV strain DHN3

<160> 66

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15192

<212> DNA

<213> Artificial sequence ()

<400> 1

accaaacaga gaatccgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaaccccgaa cgcgagatcg aagcttgaac ctgagggaac cttctaccga 120

tatgtcgtct gtttttgacg aatacgagca gctccttgct gcccagaccc gccctaacgg 180

agcccatgga gggggagaga aagggagcac cttaaaagtt gaggtcccag tatttaccct 240

aaacagtgat gatccagagg atagatggaa ctttgcggta ttctgtcttc ggattgccgt 300

tagtgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcaggaaaa cagaacgagg ccacactagc 420

tgttcttgag atcgatggtt ttgctaataa tgtgccccag ttcaacaata ggagtggagt 480

gtccgaggag agagcacaga gattcatggt aattgcaggg tctctccctc gggcatgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa aggatcctat ctgtccaagt ccaagtatgg gtcacggtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

gggtagagtc cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcat 780

aatcagacat tctctggcag tccgtatttt cctggttagt gagctcaaga ggggccgtaa 840

tacagcaggt gggagctcta catattacaa tttggtcggg gatgtagact catacatcag 900

aaataccggg cttactgcgt ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agctctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgcg ccagccgaat atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt tgccagggac tttatgagca catcattctg 1200

gcgacttgga gtagagtatg ctcaggctca gggaagtagt atcaatgaag acatggctgc 1260

tgagttaaaa ctaaccccag cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

tgaagaaatc ggcagcatgg acattcctac tcaacaagca ggagtcctca ccgggctcag 1380

tgacgaaggc ccccgaactc cacagggtgg atcgaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcctggattt tatgagaaca gtggcgaaca gcatgcggga 1500

atcgcctaat cctgcacaga gcaccactca tctagagcct cccccgaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgactact cccagcctgc ctccatagga 1620

ccacaccaaa cccctccccc aaaacccccc cacacccccc gacccacaac cccgcacgac 1680

ccccccaaca aaagctcccc cccaccctct cccccacccc cagccacacg accccatcca 1740

cccgggacaa cacaggcaca gctcggccag tcaacaatcc tcccagagtc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgggtcacc aagctctctg 1860

ttctcccttc tacccggtga gttagggtga agatggctac ctttacagat gcggagatag 1920

atgacatact tgagaccagt gggactgtca ttgatagcat aattacggcc cagggcaaat 1980

cagccgagac cgtcggaaga agtgcgatcc cgcagggcaa gaccaaagct ccaagcacag 2040

cacgggagaa gcacgggagt gcccagccac acgccagtca ggacgtcccc gaccaacaag 2100

acagaacaga aaaacagcca tccacacctg agcaggcaac cccacacaac aacccaccga 2160

ccacacccac cgaaccgccc tccacccagg ccgcaagcga gaccagcgac acacagctca 2220

aaaccggagc aagcaactcc cttctgtcca tgctcgacaa attaagcaat aaatcgtcta 2280

atgctaaaaa gggcccatgg tcgggtcccc aagaagggca tcaccaatct ccggcccaac 2340

aacacgggaa ccagtcgagc catggaagca accagggaag accacagcac caggtcaagg 2400

ccgtctctgg aaaccggggc atagacgaga acacagcata tcatggacaa cggaaggagt 2460

cacaaccatc agctggtgca acccctcatg cgccccagtc agggcagagc caagacaata 2520

ttcctgtacc tgtggatcgt gtccagctac ctgccgactt tgcgcaggcg atgatgtcta 2580

tgatggaggc attatctcag aaggtaagta aagttgatca tcagctggat ctggtcttga 2640

aacagacatc ctctattcct atgatacgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggtatgatga aaattctgga ccctggttgt gccaacattt 2760

catctttaag tgatctccgg gcagtagccc gatcccaccc agtcctagtt tcgggccccg 2820

gagacccatc tccttacgtg acacaagagg gtgaaatgac gctcaataaa ctctcacaac 2880

cagtgcagca cccttctgaa ttgattaagt ctgccaccgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcacgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca agtcaattga agagatcagg aagatcaagc 3060

gccttgcgct gaatggttga tggccgtcac aactcatagc aggctcctgt cgcttcagca 3120

tcacacggaa tcccccggga gccccccctt gcgaatccat gcttcaacac cccagacaac 3180

agccctctct caccatcccc aatccctcgc atgatcgcac aactgcaacc aatctagcag 3240

cattagagat taagaaaaaa cacgggtaga atcaaagtgc ctcgattgaa ccaaaatgga 3300

ctcatccagg acaatcgggc tgtactttga ttctgccctt ccttccagta gcctattagc 3360

atttccgatt atcttacaag atacaggaga cgggaagaaa caaatcactc cacaatacag 3420

gatccagcat cttgattcgt ggacagacag taaggaagac tcggtattta tcaccaccta 3480

cgggttcatc tttcaagttg ggaatgaagg agccactgtc ggtgtgatca atgacaatcc 3540

caggcatgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acaacggaga 3600

tcttgttgag ctggcgagag cctgcctcac tatggtggta acctgcaaga agagtgcaac 3660

taacactgag agaatagtct tctcagtagt gcaggcacct cgagtgctgc aaaattgtat 3720

ggttgtgtca aatcggtact catcagtgaa tgcagtgaag catgtgaagg cgcccgaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctccttga ctgtggtgcc 3840

gagaagggat gtctacagga tcccaactac agtattgaaa gtgtctggct cgagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttct aagtccgata gcgggtacta tgcgaatctt tttctgcata tcggacttat 4020

gtccactgta gataagagag gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatctgttg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc tttcttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaga ccgcgcacct 4260

gcggagcgtg aaagtcatca ttcaagccgg cactcagcgt gctgttgcag tgaccgccga 4320

tcatgaggta acctccacta agatagagaa gaagcatgcc attgctaaat acaatccttt 4380

cagaaaatag gttacatccc taagactgca gttcacctgc tttcccgaat catcatgaca 4440

ccagataatg atccatctcg actgcttgta gttagttcac ctgtccagca aattagaaaa 4500

aacacgggta gaagagtctg gaccccgacc ggcacactca ggacacagca tgggctccaa 4560

accttctacc aggatcccag cacctctaat gctaatcact cgaattatgc tgatattgaa 4620

ctgcatccgt ctgacaagtt ctcttgacgg caggcccctt gcagctgcag gaattgtaat 4680

aacaggagat aaggcagtca atgtatatac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gagataagga ggcatgtgca agagccccat tggaggcata 4800

taacagaaca ctgactactc tgctcactcc tcttggcgac tccattcgca agatccaagg 4860

gtctgtatcc acgtccggag gaaggagaca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctc ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caaacagaat gctgccaaca tcctccggct taaggagagc attgctgcga ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca attatcagtg gcagttggga agatgcaaca 5100

gtttgtcaat gaccagttta ataacacggc gcgagaatta gactgcataa aaatcacaca 5160

acaggtcggt gtagaactca acctatacct aactgagcta actacagtat tcgggccaca 5220

gatcacctcc cctgcattaa ctcagctaac catccaggcg ctctataatt tagctggtgg 5280

caatatggac tacttattaa ctaagttagg tataggaaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacatactc aactcttggg 5400

catacaagtt aatctgccct cagtcgggaa cttaaataat atgcgtgcca cctatctgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca ctggtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtctcca ggtatttatt cctgtttaag 5640

tggcaacaca tcagcctgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttagg ggctcagtta ttgctaattg taagataaca acatgcagat gtacagaccc 5760

tcctggcatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacactcgtg 5820

caatgtctta tcattagacg gcataactct gaggctcagt ggggaatttg atgcaactta 5880

tcaaaagaac atctcaatat tagattctca ggtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca ataattcgat cagcaatgcc ttggacaagt tggcagaaag 6000

caacagcaaa ctagaaaaag tcaacgtcag actaactagc acatccgctc tcattaccta 6060

tattgttcta actgtcattt ccctaatttt cggtgcactt agtctggttt tagcgtgtta 6120

cctgatgtac aagcagaagg cacaacaaaa gaccctgtta tggcttggga acaataccct 6180

cgatcagatg agagccacca tgagagcatg aatgcaaata ggaagtggac ggacacccaa 6240

cggcagcccg tgtgtcaatt ccgataacct gtcaagtagg agacttaaga aaaaattact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

tgaaaattag gcttcacaac atccgttcta ccacaccacc aacaacaaga gtcaatcatg 6420

gaccgcgtgg ttaacagagt catgctggag aatgaagaaa gagaagcaaa gaacacatgg 6480

cgcttagttt tccggatcgc agtcttattt ttaatggtaa tgattctagc tatctctgcg 6540

gctgccctgg catacagcat ggaggccagt acgccacacg acctcgcagg catatcgact 6600

gtgatctcca agacagaaga taaggttacg tctttactca gttcaagcca agatgtgata 6660

gataagatat acaagcaggt agctcttgaa tccccgctgg cactattaaa caccgaatct 6720

gtaattatga atgcaataac ctctctttct tatcaaatta acggggctga gaacagtagc 6780

ggatgcggtg cgcccgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca ttagtaatgt cgcatcattt tatccttctg catatcaaga acacttgaat 6900

ttcatcccgg cacctactac aggatctggt tgcactcgga taccctcatt tgacatgggc 6960

accacccatt attgttatac tcacaatgtg atactatctg gttgcagaga tcactcacac 7020

tcacatcaat acctagcact tggtgtgctt cggacatctg caacagggag ggtattcttt 7080

tctactctgc gctccatcaa tttagatgac actcaaaatc ggaagtcttg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggaggat 7200

tacaagtcag ttgcccccac atcaatggtg cacggaaggc taggatttga cggtcaatac 7260

catgagaaag acttagacac cacggtctta tttaaggatt gggtggcaaa ttacccgggg 7320

gtgggaggag ggtcttttat tgacggccgt gtatggttcc cagtttacgg agggctcaaa 7380

cctaattcac ccagtgacat cgcacaagaa gggaaatatg taatatacaa gcgccataat 7440

aacacatgcc ccgataaaca agattaccaa attcggatgg ctaagtcctc atataaacct 7500

gggcgatttg gtggaaagcg cgtacagcaa gctatcctat ctatcaaagt gtcaacatcc 7560

ctgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtatc aacgagggtc ttcatatttc 7680

tcccctgcct tattgtatcc catgacagta agtaacaaaa cggctacact ccatagtcct 7740

tacatgttta atgctttcac tcggccaggt agtgtccctt gccaggcatc agcaaggtgc 7800

cccaactcat gcatcactgg ggtctatacc gatccatatc ccttaatctt ctataggaat 7860

catactctac gaggggtctt cgggacgatg cttgatgatg aacaagcgag gcttaacccc 7920

gtatctgcag tatttgacaa catatctcgc agtcgtgtca cccgggtgag ttcaagcaac 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagactaa taaagtttat 8040

tgtcttagta tcgcagaaat atccaatacc ctattcgggg aatttaggat cgttcccttg 8100

ctagttgaga tcctcaaaga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggagga cgacattgcg ccaatcatct cccataatgc ttagagtcaa 8220

gctgaacatt agcataaatc aggatcccgt gttgttgggc aaccgcaatc cgacaatgct 8280

gacatgattg ttctgagtct cgctcactgt cactttatta agaaaaaaca caagaagcat 8340

tgacatataa gggaaaataa ccaacaagag agaacacggg taggacatgg cgggctccgg 8400

tcctgagagg gcagagcacc agatcatcct accagagtca catttatcct ctccattggt 8460

caagcacaaa ttgttatact actggaaatt aaccgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcag ccactcctga 8580

cacagagaga atgataaaac ttgggcgggc ggtgcaccag actctcaacc acaattccaa 8640

gatgactgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtccctga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caagatatgg 8760

agacctgttc acaaagctgt gcgtgcaagt tgagaagaaa ttgctagggt catctctgtc 8820

taataatgtc ccacgatcag aggaattcaa cagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ccaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taaatcataa 9000

gataggccat gtctttatta ctcctgagct tgtcattgtg acacacacag acgagaacaa 9060

gttcacatgt ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcagg aacctatccg agaaaattga 9180

tgatattctg cggttagtag atgctctggc aaaggactta ggtaatcaag tctatgatgt 9240

tgtagcatta atggagggat ttgcatacgg tgccgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaaaacacgt taatcgaact 9360

tctccccaat aatatagcgg aagcagtaac tcacgctatt gccactgtat tctctggatt 9420

agaacagaac caagcagctg agatgttgtg cttgctgcgt ttgtggggtc atccattgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgcgcaccaa agatggtaga 9540

ctttgatatg atcctccagg tattatcttt ctttaaagga acaatcatca atggatacag 9600

aaaaaagaac tcaggtgtgt ggccacgcgt caaagtagat acaatatatg gaaatatcat 9660

tgggcagcta catgctgatt cagcagagat ctcacatgat gtcatgttga gggagtacaa 9720

gagtttatcc gctcttgaat ttgagccatg tatagattat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aactggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaag gaggcaactt caactaaccg 9900

cctcctgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gagatgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtgaaa gtgaatggac ggatttttgc taaattaaca aagaaactaa ggaattgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttcttccagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagca atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatcg ctgactgcaa agagagggtt tcctcaaacc gcaatcatga 10260

tcccaagagc aagaatcgta gaagagttgc cacctttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgccatca atcagctgat 10380

gggcctacct catttctttg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gaccgactgt gatctatcaa gagtcccaaa 10500

tgatgatata tatattgtca gtgctagagg gggcattgag ggactctgtc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgccgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat agctgtaacg agagaggtga gatcagatga 10680

ttccccggat atggtattga cgcagttgca tcaggctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatctga ttggccataa cctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaagatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggtgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc ctaaggactt 10980

ctgttactat ttgaactacc taatgagttg cgtgcagaca tattttgatt cagagttttc 11040

tattactcac agctcacagt cagattccaa ccagtcctgg attgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccaact ggggggactg agtaaccttc aatactcaag 11160

gctctacaca aggaatattg gcgacccagg gaccactgcc tttgcagagg tcaagcgact 11220

agaagcagtg gggttgttga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagat tgggccagcc tatgcaacga cccatactct tttaattttg agactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccctttatta tccggggtac atacagagga caatgaggca gaagagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaagactg atgcggataa tcaattactc 11640

aagcatgcat gcaatgttgt tcagggatga tattttctta tccactagat ccaaccaccc 11700

attagtttct tctaatatgt gctcgctgac gctagcagat tatgctcgga acagaagctg 11760

gtcacccctg acagggggca ggaaaatact gggtgtatcc aaccccgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagttg actgatgaca ccagcaaaaa 11940

tcccccgatg agagtgccat atctcgggtc gaagactcaa gagagaagag ccgcctcact 12000

tgcgaaaata gcccatatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca atgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtatcttcg gctattgtca cccctgccca cagctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacattc acatatccaa tgattctcaa aggctgttca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccagca aattatgctc ttgggtttat ctctaattga 12360

gtcactcttc ccaatgacaa caaccagaac atacgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgtcgcagtt cctttcgagc tcctcggact 12480

ggtaccggaa ttaaggatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagggat tttgcgagac ttgacttagc tatattcaag agttatgagc ttaacttgga 12600

atcatatccc acgctggagc taatgaacat tctttcgata tctagcggga aattgattgg 12660

ccaatctgtg gtttcttatg atgaagatac ttctataaag aatgatgcta taatagtgta 12720

tgacaacaca cggaattgga ttagtgaggc acagaactca gatgtggtcc gcctgtttga 12780

gtatgcagca ctcgaagtgc tcctcgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcagcca cgatatccca ccccctcatt cactcaaggt tgaatgcagt 12960

aggtctaatt aatcatgacg ggtcacacca gcttgcagat atagactttg tcgaggtgtc 13020

tgcgaaattg ttagtctcct gcactcgacg cgtggtctca ggtttatatg cagggagtaa 13080

gtatgatctg ctgtttccat ctgtcttaga tgataacctg aatgagaaga tgcttcaact 13140

aatttcccgg ttatgctgct tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tattgtcgga 13260

tgctgtaaaa ccgttgctta ggtccgaaca attgagttct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat actacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcgttgtt gttccctcag gagtcactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccgacaac ccgcatcttt 13500

catacaagag ctggatctga gtgccccagc aaggtacgac gcgtttacac tgagtaagat 13560

ttgcttcgag cacacactac cgaacccaag ggaagattac ctagtacgat acttgttcag 13620

aggagtaggg actgcttcat cttcttggta taaggcgtct catcttctat ccatatctga 13680

ggttaggtgt gcaagacatg ggaactcttt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gaccatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc ggcatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttcta 13920

tccattatgg agagagaatg cagaagaaag tgatctgacc tcagataagg cagttggata 13980

tatcacatct gtagtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc agtcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg agagagggcg gggtagtgat catcaaggta ctgtatgcaa tggggtacta 14160

cttccactta ctcatgaatt tattcactcc atgttccacg aaaggataca tactttccaa 14220

tggctacgcc tgtagagggg atatggagtg ttacctgata ttcgttatgg gctgcttagg 14280

cgggcccact ttcgtgcacg aagtggtaag gatggcaaaa gctctaatac aacgacacgg 14340

tacacttcta tctaaatcag atgaaatcac attgactaag ctatttacct cacagcagcg 14400

tcgtgtaaca gatctcctat ccagcccttt accgaagcta atgaggctct taagtgaaaa 14460

cattgatgct gcactaattg aagccggggg acagcccgtc cgtccatttt gtgcagaaag 14520

tttggtgagc acactaacaa atacgaccca gacaactctg atcattgcca gccacattga 14580

cacagtcatc cggtccgtga tttacatgga ggctgagggt gacctcgccg acacagtgtt 14640

cttattaact ccttacaatc tatccacaga cggtaaaaag agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatatt gggtctcaga gccaaagaca tcaataaaat 14760

aggtgatgta atcagcttag tactcagagg tgcgatttcc ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcaca gtacctgtcc taaatacctg aaagcggtcc taggtattac 14880

taagctcaaa gaaatgttca caggtacttc gttattgtac ttgactcgcg ctcaacaaaa 14940

attctacatg aaaactatag gtaatgctgc caagggatat tacagtaata atgactctta 15000

aaggcaatcg tacaccaatc agttatcttc ttaactgatg actccctcat tgacttgatt 15060

ataccagatt agaaaaaagt taaattctga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaaaaatg cgcaaagtcg tctctaatca cagctatgtc attcaccaaa 15180

tctctgtttg gt 15192

<210> 2

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 2

ggatggttgg gaggacgac 19

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 3

gcaactgcgt caacaccat 19

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 4

gttagcaatg agtcaactgt cc 22

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 5

cttcttcggt gaacagcctt tg 22

<210> 6

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 6

ctacagagtg tcgccttac 19

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 7

ggtaaagggc tggataggag a 21

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 8

tctccaatgg ctatgcctgt a 21

<210> 9

<211> 17

<212> DNA

<213> Artificial sequence ()

<400> 9

tgggagtgga gaaggac 17

<210> 10

<211> 17

<212> DNA

<213> Artificial sequence ()

<400> 10

gttatctgtg ccgctgt 17

<210> 11

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 11

gcgcaccaaa cagagatttg gtgaatgac 29

<210> 12

<211> 16

<212> DNA

<213> Artificial sequence ()

<400> 12

gactccattc gcaaga 16

<210> 13

<211> 16

<212> DNA

<213> Artificial sequence ()

<400> 13

cattctccag cacgac 16

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 14

ccgataacct gtcaagtag 19

<210> 15

<211> 17

<212> DNA

<213> Artificial sequence ()

<400> 15

gtcagcattg tcggatt 17

<210> 16

<211> 16

<212> DNA

<213> Artificial sequence ()

<400> 16

gaacaagccg caaggg 16

<210> 17

<211> 32

<212> DNA

<213> Artificial sequence ()

<400> 17

catctgcaga cagtcccact ggtctcaagt at 32

<210> 18

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 18

catacttgag accagtggga ctgtc 25

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 19

caggagcctg ctatgagt 18

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 20

accaaacaga gaatccgtga ggta 24

<210> 21

<211> 17

<212> DNA

<213> Artificial sequence ()

<400> 21

tcagtacccc cagtcag 17

<210> 22

<211> 17

<212> DNA

<213> Artificial sequence ()

<400> 22

gtaaaacgac ggccagt 17

<210> 23

<211> 17

<212> DNA

<213> Artificial sequence ()

<400> 23

caggaaacag ctatgac 17

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 24

gcaacgtgct ggttattgtg 20

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 25

ggacagttga ctcattgcta acata 25

<210> 26

<211> 49

<212> DNA

<213> Artificial sequence ()

<400> 26

actcactata gggcgaattc ggatccggat ggttgggagg acgacattg 49

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 27

tatgttagca atgagtcaac tgtcc 25

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 28

gtgaatgtaa ggcgacactc tgtag 25

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 29

ctacagagtg tcgccttaca ttcac 25

<210> 30

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 30

cgaatatcag gtaacactcc atatc 25

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 31

gatatggagt gttacctgat attcg 25

<210> 32

<211> 48

<212> DNA

<213> Artificial sequence ()

<400> 32

taagatctgg taccgagctc ctgcaggcgc accaaacaga gatttggt 48

<210> 33

<211> 40

<212> DNA

<213> Artificial sequence ()

<400> 33

atatgaattc gccaccatgg ctacctttac agatgcggag 40

<210> 34

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 34

tatactcgag tcaaccattc agcgcaagg 29

<210> 35

<211> 44

<212> DNA

<213> Artificial sequence ()

<400> 35

accggaattc gccaccatgt cgtctgtttt tgacgaatac gagc 44

<210> 36

<211> 32

<212> DNA

<213> Artificial sequence ()

<400> 36

atatctcgag tcagtacccc cagtcagtgt cg 32

<210> 37

<211> 71

<212> DNA

<213> Artificial sequence ()

<400> 37

attgcatcaa cgcatatagc gctagcgcga tgtaccgcgg atcgttacca aacagagaat 60

ccgtgaggta c 71

<210> 38

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 38

catctggttg cccttgcggc ttgttc 26

<210> 39

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 39

gaacaagccg caagggcaac cagatg 26

<210> 40

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 40

gacagtccca ctggtctcaa gtatg 25

<210> 41

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 41

catacttgag accagtggga ctgtc 25

<210> 42

<211> 52

<212> DNA

<213> Artificial sequence ()

<400> 42

tgtttgacag cttatcatcg ataagcttcc tccatcatag acatcatcgc ct 52

<210> 43

<211> 50

<212> DNA

<213> Artificial sequence ()

<400> 43

attgcatcaa cgcatatagc gctagcaggc gatgatgtct atgatggagg 50

<210> 44

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 44

gacagtgtcc ttctccactc ccatg 25

<210> 45

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 45

catgggagtg gagaaggaca ctgtc 25

<210> 46

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 46

gacccttgga tcttgcgaat ggagtc 26

<210> 47

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 47

gactccattc gcaagatcca agggtc 26

<210> 48

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 48

gtctcctact tgacaggtta tcgg 24

<210> 49

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 49

ccgataacct gtcaagtagg agac 24

<210> 50

<211> 53

<212> DNA

<213> Artificial sequence ()

<400> 50

tgtttgacag cttatcatcg ataagcttgt gcgatgtcac tgggtgaatt agg 53

<210> 51

<211> 51

<212> DNA

<213> Artificial sequence ()

<400> 51

attgcatcaa cgcatatagc gctagcccta attcacccag tgacatcgca c 51

<210> 52

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 52

cgcaatgtcg tcctcccaac catcc 25

<210> 53

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 53

ggatggttgg gaggacgaca ttgcg 25

<210> 54

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 54

gtgaatgtaa ggcgacactc tgtag 25

<210> 55

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 55

ctacagagtg tcgccttaca ttcac 25

<210> 56

<211> 89

<212> DNA

<213> Artificial sequence ()

<400> 56

ttgacagctt atcatcgata agcttgcgat gagcggccgc tccattaata cgactcacta 60

taggaccaaa cagagatttg gtgaatgac 89

<210> 57

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 57

atcggtagaa ggttccctca ggttc 25

<210> 58

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 58

ggtcctatag tgagtcgtat taatg 25

<210> 59

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 59

gaaattgcat caacgcatat agcgc 25

<210> 60

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 60

catctggttg cccttgcggc ttgttc 26

<210> 61

<211> 49

<212> DNA

<213> Artificial sequence ()

<400> 61

actcactata gggcgaattc ggatccgcca tgaacacgat taacatcgc 49

<210> 62

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 62

gaagtccgac tctaagatgt cacg 24

<210> 63

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 63

ccttgcagct gcaggaattg 20

<210> 64

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 64

gctctataca gtatgaggtg tcaag 25

<210> 65

<211> 50

<212> DNA

<213> Artificial sequence ()

<400> 65

taagatctgg taccgagctc ctgcagttac gcgaacgcga agtccgactc 50

<210> 66

<211> 4306

<212> DNA

<213> Artificial sequence ()

<400> 66

gtgagtttgg ggacccttga ttgttctttc tttttcgcta ttgtaaaatt catgttatat 60

ggagggggca aagttttcag ggtgttgttt agaatgggaa gatgtccctt gtatcaccat 120

ggaccctcat gataattttg tttctttcac tttctactct gttgacaacc attgtctcct 180

cttattttct tttcattttc tgtaactttt tcgttaaact ttagcttgca tttgtaacga 240

atttttaaat tcacttttgt ttatttgtca gattgtaagt actttctcta atcacttttt 300

tttcaaggca atcagggtat attatattgt acttcagcac agttttagag aacaattgtt 360

ataattaaat gataaggtag aatatttctg catataaatt ctggctggcg tggaaatatt 420

cttattggta gaaacaacta catcctggtc atcatcctgc ctttctcttt atggttacaa 480

tgatatacac tgtttgagat gaggataaaa tactctgagt ccaaaccggg cccctctgct 540

aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg 600

ctgtctcatc attttggcaa agaattgtaa tacgactcac tatagggcga attcggatcc 660

aagcttctcg aggcggccgc cccgggctgc aggagctcgg taccagatct tattaaagca 720

gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 780

aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc 840

ttatcatgtc tggtcgactc tagactcttc cgcttcctcg ctcactgact cgctgcgctc 900

ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 960

agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 1020

ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 1080

caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 1140

gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 1200

cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 1260

tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 1320

gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 1380

cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 1440

tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 1500

tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 1560

caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 1620

aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 1680

cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 1740

ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 1800

tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 1860

atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 1920

tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 1980

aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 2040

catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 2100

gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 2160

ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 2220

aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 2280

atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 2340

cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 2400

gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 2460

agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 2520

gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 2580

caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 2640

ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 2700

tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 2760

aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat 2820

catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg 2880

tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 2940

agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 3000

gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg 3060

tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat 3120

attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc 3180

gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt 3240

ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa 3300

accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg 3360

tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga 3420

cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 3480

agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat 3540

gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat 3600

cggtgcgggc ctcttcgcta ttacgccagc tagagtccgt tacataactt acggtaaatg 3660

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 3720

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 3780

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 3840

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 3900

cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 3960

acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 4020

acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 4080

actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 4140

gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc 4200

atagaagaca ccgggaccga tccagcctcc gcggccggga acggtgcatt ggaacggacc 4260

cctaggcttt tgcaaaaagc tccggatcga tcctgagaac ttcagg 4306

Claims (3)

1. A recombinant cloning method aiming at II type VII epidemic NDV strain DHN3 is characterized by comprising the following steps:

step 1: constructing helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L, wherein target fragments of the helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L are NP gene, P gene and L gene respectively, and a vector is pXJ 40;

step 2: constructing a whole genome expression vector pBR322-DHN 3: the DHN3 whole genome obtained by artificial recombination is recombined into pBR322 plasmid to obtain a whole genome expression vector pBR322-DHN 3; the DHN3 whole genome is the whole genome of II-type VII-type newcastle disease virus; the whole genome sequence of DHN3 is shown in a sequence table SEQ ID NO 1;

and step 3: co-transfecting helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L and a whole genome expression vector pBR322-DHN3 to BHK-21 cells to obtain virus liquid containing recombinant virus rDNN 3;

wherein, the step 2 specifically comprises the following steps:

step 21: establishing a pBR322-Base vector: introducing a fragment capable of homologous recombination with the whole genome of DHN3 into a pBR322 plasmid; the fragment has homology arms corresponding to the 3 'end and the 5' end of the DHN3 whole genome;

step 22: constructing a transition vector: the transition vector is plasmid pBR322-PNP, plasmid pBR322-PDP and plasmid pBR322-LPD 3;

from 5 'end to 3' end, the sequence of the target fragment of the plasmid pBR322-PNP is NP, MINI and P gene sequence; from 5 'end to 3' end, the target fragment sequence of the plasmid pBR322-PDP is P, PD1, PD2 and PD3 gene sequences; from 5 'end to 3' end, the target fragment sequence of plasmid pBR322-LPD3 is PD3, L1, L2, L3 and L4 gene sequence;

the preparation methods of the plasmid pBR322-PNP, the plasmid pBR322-PDP and the plasmid pBR322-LPD3 are as follows:

A. preparation of plasmid fragment: performing double enzyme digestion on the pBR322 vector by using Hind III and Nhe I, and then recovering the pBR322 plasmid fragment for later use by using glue;

B. preparing a target gene fragment, wherein the target gene fragment is NP, MINI, P, PD1, PD2, PD3, L1, L2, L3 and L4 genes;

C. connecting a pBR322 plasmid fragment target gene fragment with a corresponding target gene fragment by using a recombinase, performing bacterial transformation, performing single colony PCR (polymerase chain reaction) primary selection, amplifying plasmid DNA, performing DNA glue check after the plasmid DNA is subjected to enzyme digestion, and finally performing sequencing verification to obtain a plasmid pBR322-PNP, a plasmid pBR322-PDP and a plasmid pBR322-LPD 3;

step 23: construction of DHN3 Whole genome DHN 3-A: plasmid pBR322-PDP and plasmid pBR322-LPD3 are digested separately with BtgZ I to recover gene fragment PDP and LPD 3; the vector pBR322-PNP is subjected to double enzyme digestion by BtgZ I and Hind III to recover a gene fragment PNP; carrying out in vitro connection on the gene fragment PNP, the gene fragment PDP and the gene fragment LPD3 through T4 ligase to obtain DHN3 whole genome DHN 3-A;

step 24: constructing a plasmid fragment with a homology arm: carrying out PCR amplification by taking the pBR322-Base vector in the step 21 as a template to obtain a plasmid fragment with a homology arm;

step 25: constructing a whole genome expression vector pBR322-DHN 3: carrying out homologous recombination on the plasmid fragment with the homologous arm in the step 24 and the DHN3 whole genome DHN3-A in the step 23 to obtain a plasmid pBR322-DHN3 with the DHN3 whole genome DHN 3-A;

wherein, the NP gene is shown in a sequence table SEQ ID NO: the 1 middle position is 1-1591 nt; the P gene is shown in a sequence table SEQ ID NO: the position in the 1 is 1925 and 3109 nt; the L gene is shown in a sequence table SEQ ID NO: the 1 position is 8166 + 15192 nt; the MINI gene is shown in a sequence table SEQ ID NO: the 1 position is 1414-; the PD1 gene is shown in a sequence table SEQ ID NO: the 1-position is 2935-; the PD2 gene is shown in a sequence table SEQ ID NO: the 1 middle position is 4838 and 6454 nt; the PD3 gene is shown in a sequence table SEQ ID NO: the 1 position is 6261 and 8283 nt; the L1 gene is shown in a sequence table SEQ ID NO: the position in the 1 position is 8166 + 10709 nt; the L2 gene is shown in a sequence table SEQ ID NO: the 1-position is 10174 and 12299 nt; the L3 gene is shown in a sequence table SEQ ID NO: the 1-position is 12238-; the L4 gene is shown in a sequence table SEQ ID NO: position 14214- > 15192nt in 1.

2. The method for recombinantly cloning a class II type VII epidemic NDV strain DHN3 according to claim 1, wherein step 3 specifically comprises: co-transfecting BHK-21 cells with plasmids capable of expressing T7RNA polymerase, helper plasmids pXJ40-NP, pXJ40-P, pXJ40-L and a whole genome expression vector pBR322-DHN 3; obtain virus liquid containing recombinant virus rDNN 3.

3. The method for recombinantly cloning a class II type VII epidemic NDV strain DHN3 according to claim 1, wherein step 21 specifically comprises: introducing 1T 7 promoter, 1T 7 terminator and 1 HDV Ribozyme into pBR322 plasmid; partial base HC1 introduced to the 3' end of DHN3 downstream of the T7 promoter; the base HC2 in the 5' terminal part of DHN3 was introduced upstream of HDV Ribozyme; wherein, the position sequence of the base HC1 in the DHN3 whole genome sequence is 15192-15159 nt; the base HC2 has a position sequence of 141-1nt in the whole genome sequence of DHN 3.

Images