CN109402071B

CN109402071B - Recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein

Info

Publication number: CN109402071B
Application number: CN201811324183.7A
Authority: CN
Inventors: 刘金华; 刘立涛; 孙洪磊
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-11-08
Filing date: 2018-11-08
Publication date: 2021-04-27
Anticipated expiration: 2038-11-08
Also published as: CN109402071A

Abstract

The invention discloses a recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein. The recombinant herpesvirus of turkeys is obtained by modifying the genome of the original herpesvirus of turkeys as follows: the DNA molecule of the coding gene containing H9N2 subtype avian influenza virus H9 protein is increased; the amino acid sequence of the H9N2 subtype avian influenza virus H9 protein is shown as a sequence 2in a sequence table. Experiments prove that on the 1 st day of the experiment, the neck of the SPF chicken aged 1 day is inoculated with the recombinant turkey herpesvirus subcutaneously; experiment day 35, nasal inoculation with BJ/15 strain; on days 38 and 40 of the experiment, the oral cavity and cloaca of the SPF chicken are not detoxified, and the BJ/15 virus strain cannot replicate effectively in the viscera (such as internal organs, trachea, lung, spleen and kidney) of the SPF chicken. Therefore, the recombinant herpesvirus of turkeys has excellent immunoprotection effect against AIV subtype H9N 2. The invention has important application value.

Description

Recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein.

Background

In 1994, the situation that chickens are infected with H9N2 subtype Avian Influenza Virus (AIV) is firstly reported in China, and since the H9N2 subtype AIV is widely popularized in domestic poultry, the virus causes egg laying of laying hens to be reduced or secondary infection after infecting a chicken group, and huge economic loss is brought to the breeding industry. H9N2 subtype AIV is popular in terrestrial birds and evolves into different genotypes, the dominant epidemic strain of H9N2 subtype AIV in chicken flocks in China is G57 genotype at present, the genotype appears in 2007 at the earliest, and becomes the dominant genotype after 2010; the H9N2 subtype AIV of the genotype not only seriously threatens the healthy development of the breeding industry, but also plays an important role in the generation of novel influenza viruses such as H7N9, H10N8 and the like as a gene donor, and brings great challenges to the prevention and control of avian influenza viruses.

Aiming at AIV of H9N2 subtype, China implemented the immune control strategy of inactivated vaccine since 1998. However, the inactivated vaccine has many inherent defects, which can only stimulate the organism to generate humoral immunity but not effective cellular immunity, and meanwhile, the immune protection generated by the inactivated vaccine is mainly achieved through the high serum antibody level, because the antibody generated by the inactivated vaccine is short in maintenance time, the continuous multiple immunization is needed to maintain the high antibody titer, so that huge workload is brought to the cultured people, and meanwhile, the excessive immunization causes the organism immune fatigue, the immune effect is reduced, and the production performance is further reduced. The inactivated vaccine takes longer time to generate the antibody, and the optimal protection time is easy to miss. Therefore, in the past 20 years, the inactivated vaccine can relieve the clinical symptoms of infected chickens, reduce the detoxification of the chickens, but cannot produce complete protection, and simultaneously, the epidemic of H9N2 subtype AIV cannot be effectively controlled due to the variation of virus antigenicity. In recent years, the prevalence of H9N2 subtype AIV in immune chicken flocks is investigated, and the separation rate of H9N2 subtype AIV is increased year by year from 22.08% in 2010 to 47.08% in 2013, which indicates that the traditional inactivated vaccine cannot effectively prevent and control H9N2 subtype AIV infection, so that the development of a new vaccine is imminent.

The live vector vaccine can stimulate the organism to generate humoral immunity and cellular immunity, and becomes a hotspot for developing new vaccines. Herpesvirus of turkeys (HVT) belongs to the family herpesviridae and has been widely used as a vaccine strain for the immune control of Marek's Disease (MD) since its isolation in the 1970 s. HVT can contain and carry exogenous genes for replication, and is the hot door of live vector vaccine research at present. Since 1970, HVT FC126 strain has become the major vaccine for MD prevention worldwide. HVT is double-stranded DNA virus with large genome and many replication nonessential regions, and can carry foreign gene to replicate and express in vivo. Simultaneously still have following advantage: HVT can continuously infect in the chicken body, stimulate the organism to continuously generate antibodies, and the effect of lifetime immunity can be achieved by inoculating once; HVT as vaccine has no pathogenicity to chicken, does not influence the production performance, and has good safety; HVT can stimulate an organism to generate stronger cellular immune response, has long duration, and can achieve the effect of lifetime immunity after being inoculated once; HVT has strong cell binding property, can be spread among cells, and breaks through the interference of maternal antibodies and the like. Therefore, the genetic engineering vaccine taking HVT as the vector has wide application prospect. Currently, the HVT is used as a vector to successfully express immunoprotective antigens of a plurality of avian viruses, such as Infectious Bursal Disease Virus (IBDV), infectious laryngotracheitis virus (ILTV), Newcastle Disease Virus (NDV), Eimeria and the like, and the HVT recombinant vaccines can not only simultaneously induce the avian to generate immunoprotection against viral vectors and foreign viruses and play a role in preventing various diseases, but also have long-lasting and short-latency immunoreaction generated in vivo by the recombinant viruses.

Disclosure of Invention

The invention aims to prepare a vaccine for preventing avian influenza virus and Marek's virus.

The invention firstly protects a recombinant turkey herpesvirus; the recombinant herpesvirus of turkeys is obtained by modifying the genome of the original herpesvirus of turkeys as follows: the DNA molecule of the coding gene containing H9N2 subtype avian influenza virus H9 protein is increased;

the H9N2 subtype avian influenza virus H9 protein can be a1) or a2) or a3) as follows:

a1) the amino acid sequence is protein shown as a sequence 2in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2in the sequence table;

a3) the protein obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a2) and having the same function.

The encoding gene of the H9N2 subtype avian influenza virus H9 protein can be c1) or c2) or c3) as follows:

c1) a DNA molecule shown in a sequence 1 of a sequence table;

c2) a DNA molecule having more than 75% identity with the DNA molecule defined by c1) and encoding the H9N2 subtype avian influenza virus H9 protein;

c3) a DNA molecule or a cDNA molecule which is hybridized with the DNA molecule defined by c1) or c2) under strict conditions and encodes the H9N2 subtype avian influenza virus H9 protein.

The H9N2 subtype avian influenza virus H9 protein is derived from an avian influenza virus strain named A/Chicken/Beijing/0701/2015(H9N2), which is specifically isolated and identified by the method described in the reference (Pu juan et al. evaluation of the H9N2 underfluenza genetic type at deteriorated the genetic information of the novel H7N9virus.Proc Natl Acad Sci U S A.2015.) by collecting a throat swab sample of Chicken from the live bird market in Beijing by the inventors of the present invention.

In any of the above recombinant herpesviruses of turkeys, the mode of implementation of the transformation may be: inserting specific DNA molecules between 95322nt and 95323nt of the UL region of the genome of the herpes virus of the starting turkey; the specific DNA molecule can contain a promoter, a coding gene of the H9N2 subtype avian influenza virus H9 protein and a terminator.

The specific DNA molecule also can contain an enhancer; the enhancer may be located upstream of the promoter.

Any one of the specific DNA molecules can sequentially contain the enhancer, the promoter, the coding gene of the H9N2 subtype avian influenza virus H9 protein and the terminator from upstream to downstream.

Any of the enhancers described above may be a CMV enhancer. The CMV enhancer may be a DNA molecule represented by positions 1 to 274 from the 5' end of the sequence 3 in the sequence list.

Any of the promoters described above may be a chicken β -actin promoter. The chicken beta-actin promoter can be a DNA molecule shown in 275 th to 561 th positions from the 5' end of a sequence 3 in a sequence table.

Any of the above terminators may be a bGH terminator. The bGH terminator can be a DNA molecule shown in 2350-2573 th site from the 5' tail end of a sequence 3 in a sequence table.

Any one of the specific DNA molecules can be a DNA molecule shown in a sequence 3 of a sequence table.

Any of the herpes viruses of the starting turkeys can be specifically an HVT FC-126 vaccine strain.

Any of the specific DNA molecules described above also fall within the scope of protection of the present invention.

Any H9N2 subtype avian influenza virus H9 protein also belongs to the protection scope of the invention.

Any of the encoding genes of the H9N2 subtype avian influenza virus H9 protein also belongs to the protection scope of the invention.

The invention also protects X1) or X2) or X3) or X4).

X1) the application of any recombinant herpesvirus of turkeys in preventing avian influenza virus and/or Marek's virus also belongs to the protection scope of the present invention.

X2) the application of any recombinant turkey herpesvirus in the preparation of a vaccine for preventing avian influenza virus and/or Marek's virus also belongs to the protection scope of the invention.

X3) any one of the H9N2 subtype avian influenza virus H9 protein, or any one of the H9N2 subtype avian influenza virus H9 protein coding gene, or any one of the specific DNA molecules in the application of preventing avian influenza virus also belongs to the protection scope of the invention.

X4) any one of the H9N2 subtype avian influenza virus H9 protein, or any one of the H9N2 subtype avian influenza virus H9 protein coding gene, or any one of the specific DNA molecules in the preparation of vaccines for preventing avian influenza virus also belong to the protection scope of the invention.

The invention also provides a vaccine for preventing avian influenza virus and/or Marek's virus, which can contain any of the recombinant herpesviruses of turkeys.

The vaccine for preventing avian influenza virus and/or Marek's virus is prepared by taking any recombinant herpesvirus of turkeys as an antigen.

Any one of the avian influenza viruses can be H9N2 subtype avian influenza virus.

Experiments prove that on the 1 st day of the experiment, the neck of the SPF chicken aged 1 day is subcutaneously inoculated with the recombinant gene provided by the inventionTurkey herpesvirus, the inoculation dose is 3000 PFU/mouse; on day 35 of the experiment, the nasal cavity was inoculated with BJ/15 strain at a dose of 10⁴ ELD 50; on days 38 (challenge day 3) and 40 (challenge day 5) of the experiment, both the oral cavity and cloaca of SPF chickens were not detoxified, and the BJ/15 strain was not able to replicate efficiently in the internal organs (e.g., internal organs, trachea, lungs, spleen and kidneys) of SPF chickens. Therefore, the recombinant herpesvirus of turkeys provided by the present invention has excellent immune protective effect on H9N2 subtype AIV. The invention has important application value.

Drawings

Fig. 1 is a phylogenetic tree drawn from the HA gene of H9N2 avian influenza virus.

FIG. 2 is an antigen profile prepared based on the results of hemagglutination inhibition assays.

FIG. 3 is a schematic diagram of the construction of SW102 strain containing the recombinant plasmid HVT-BAC-H9.

FIG. 4 is a schematic diagram of the construction of recombinant virus rHVT-H9.

FIG. 5 shows the results of indirect immunofluorescence assay.

FIG. 6 shows the result of Western blot identification.

FIG. 7 shows the results of serum HI antibody titer assays.

Fig. 8 is the serum VN antibody titer test results.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The pcDNA3.1+ vector is a product of Invitrogen corporation under the catalog number V790-20. The pBeloBAC11 plasmid and the pUC19 plasmid were both products of NEB corporation under the catalog numbers E4154 and N3041S, respectively.

The HVT FC-126 vaccine strain is maintained at the university of agriculture in China, and is publicly available at the university of agriculture in China (i.e., at the applicant's location). In the following examples, HVT FC-126 vaccine strain is abbreviated as HVT.

The nucleotide sequences of the primers referred to in the examples below are specifically shown in Table 1.

TABLE 1 primer names and sequences required for construction

Example 1 acquisition and identification of A/Chicken/Beijing/0701/2015(H9N2) (hereinafter referred to as A/Chicken/Beijing/0701/2015, BJ/15 Strain or CK/BJ/1/15)

Isolation and characterization of H9N2influenza Virus

The inventor of the present invention collected throat swab samples of chicken from Beijing, Tianjin, Hebei and Shandong, etc. in China between 2014 and 2015, and then isolated and identified the relevant viruses of the samples by the method described in the reference (Pu juan et al. evolution of the H9N2 underfluenza genetic analysis of the novel H7N9virus.Proc Natl Acad Sci U S.A.2015.). 16 strains of H9N2 avian influenza virus were isolated. According to the general nomenclature of influenza viruses, 16 strains of H9N2 avian influenza viruses obtained by separation are named, and the specific information is shown in Table 1-1.

TABLE 1-1.2014-2015 basic information for H9N2 avian influenza virus isolated

The following examples also used the laboratory-preserved avian influenza virus of the present invention, isolated from 10 strains H9N2 before 2014, and the specific information is shown in tables 1-2. These 10 strains of H9N2 avian influenza virus are described in the following documents: qinxinghua, 2016.2014-2015, gene evolution and antigenicity analysis of H9N2influenza virus of poultry in northern region of China.

TABLE 1-2 basic information of H9N2 avian influenza virus preserved in inventors' laboratories

Serial number	Strain name	For short	Time of separation	Separation site
					1	A/quail/Hong Kong/G1/97	G1	1997	Hong Kong
2	A/Chicken/Beijing/3/99	3/99	1999	Beijing
					3	A/Chicken/Shandong/ZB/07	ZB/07	2007	Shandong (mountain east)
4	A/Chicken/Guangdong/01/11	GD/01/11	2011	Guangdong (Chinese character of Guangdong)
					5	A/Chicken/Hebei/0617/07	0617/07	2007	Hebei river
6	A/Chicken/HeB/YT/10	YT/10	2010	Hebei river
					7	A/Chicken/Shandong/qd1115/12	SD/1115/12	2012	Shandong (mountain east)
8	A/Chicken/Shandong/01/10	SD/01/10	2010	Shandong (mountain east)
					9	A/Chicken/Jiangsu/TS/10	TS/10	2010	Jiangsu
10	A/Chicken/Shandong/06/11	SD/06/11	2011	Shandong (mountain east)

Second, H9N2 avian influenza virus phylogenetic relationship analysis

In order to systematically study the gene evolution of the isolated 16 strains of H9N2 avian influenza virus, the inventors of the present invention downloaded representative strains of each branch of the trees of various gene fragments of H9N2 avian influenza virus in the literature in the Nucleotide of NCBI database according to the analysis method of the phylogenetic relationship in the literature (Pu J, Wang S, Yin Y, Zhang G, Carter R A, Wang J, et al. evolution of the H9N2influenza viral at the faulted production of the novel H7N9 viruses. Proc Natl Acad Sci U A2015; 112: 548 553); additionally, sequences of H9N2 avian Influenza virus published between 2014 and 2015 two years were downloaded in infiluenza of NCBI database, and phylogenetic trees were drawn together with sequencing results of 16 strains of H9N2 avian Influenza virus isolated in step one. The method for drawing the phylogenetic tree comprises the following steps: firstly, using MEGA6 software to compare all sequences, storing the comparison result, translating the corresponding nucleotide sequence into the corresponding amino acid sequence, and carrying out corresponding statistics and analysis; then selecting Neighbor-Joining Tree in Phylogeny, and making the evolutionary Tree by using other parameters as default values.

The phylogenetic tree plotted against the HA genes is shown in FIG. 1. The result shows that the HA gene of the H9N2 avian influenza virus can be divided into 10 large branches, and the HA genes of the 16 strains of H9N2 avian influenza virus separated in the first step belong to the Major group subbranch of the clade 9 branch. In the 16 strains of H9N2 avian influenza virus separated in the step one, the gene evolution relationship of the separated strains in 2014 is closer to that of other strains; the gene evolution relation of H9N2 avian influenza virus isolated in 2015 is relatively close. Overall, the H9N2 avian influenza virus that has been reported and isolated in step one after 2014 (including 2014, and a few strains in 2013) forms a new sub-branch within the clade 9 branch. After alignment by the Neighbor-Joining method of MegAlign software, the nucleic acid sequence homology of the HA gene of the 16H 9N2 avian influenza viruses isolated in step one was 96.3% to 99.9%.

Antigen analysis of H9N2 avian influenza virus

1. Preparation of antiserum

And (3) preparing antiserum by taking the 16 strains of H9N2 avian influenza virus separated in the step one and 10 strains of H9N2 avian influenza virus shown in the table 1-2. The methods for preparing antisera are referenced in the following documents: qinxinghua, 2016.2014-2015, gene evolution and antigenicity analysis of H9N2influenza virus of poultry in northern region of China.

2. Hemagglutination inhibition assay

And (3) taking the antiserum prepared in the step (1) and the H9N2 avian influenza virus thereof to perform a hemagglutination inhibition test. Specific experimental methods and procedures are referenced in the following documents: (Cox N, Webster R G, Krauss S, Guan Y, Hay A, Yu K, et al. WHO Manual on Animal Influenza Diagnosis and Surveillance,2nd edition.2005).

The results are shown in Table 2. The result shows that the corresponding serum hemagglutination inhibition titer of the antigen group F of the H9N2 avian influenza virus and the antigen group A, C, E is low, and the antigen cross reaction is poor; and the serum hemagglutination inhibition titer is higher and the antigen cross reaction is better corresponding to the antigen group D, F. Among the 16 strains of H9N2 avian influenza virus separated in the step one, the serum corresponding to the antigen group A, C, E has lower hemagglutination inhibition titer and poorer antigen cross reaction except individual strains of the H9N2 avian influenza virus separated in 2014; the H9N2 avian influenza virus isolated from 2015 has lower hemagglutination inhibition titer on serum corresponding to A, C, D, E, F and poorer antigen cross reaction, and the hemagglutination inhibition titer of antiserum corresponding to only part of strains of the antigen group D, F1 is higher and the antigen cross reaction is better. Antiserum corresponding to the H9N2 virus strain separated in 2014 and 2015 has lower hemagglutination inhibition titer and poorer antigen cross reaction when reacting with the strain of the antigen group A, C, D, E, F; and has higher hemagglutination inhibition titer and better antigen cross reaction with the H9N2 avian influenza virus in 2015. Combining the above results, it can be concluded that avian influenza virus H9N2 isolated in 2015 forms a new antigen group G.

TABLE 2-1 hemagglutination inhibition assay for H9N2 avian influenza virus antigenicity Change results

Note: < indicates that the antibody titer was less than 16.

TABLE 2-2 hemagglutination inhibition assay for H9N2 avian influenza virus antigenicity Change results

Note: < indicates that the antibody titer was less than 16.

(ii) the results of the hemagglutination inhibition assay are presented on the website: (http://sysbio.cvm.msstate.edu/AntigenMap) Making corresponding antigen map. The antigenic profile is shown in FIG. 2. The results indicate that the isolation of avian influenza virus H9N2in 2015 formed a new antigen group, designated G, which did not cross other antigen groups.

3. Micro neutralization test

Taking the antiserum prepared in the step 1 and the H9N2 avian influenza virus thereof, and carrying out a micro-neutralization test on MDCK cells.

The results are shown in Table 3. The result shows that the neutralizing titer of the serum corresponding to the antigen group F and the antigen group A, C, E is low, and the antigen cross reaction is poor; and the neutralizing titer of the serum corresponding to the antigen group D, F is higher, and the antigen cross reaction is better. The antiserum corresponding to the H9N2 avian influenza virus separated in 2015 has low neutralizing titer with an antigen group A, C, D, E, F, and has poor antigen cross reaction; similarly, the H9N2 avian influenza virus isolated in 2015 had low neutralization titers as did the antisera corresponding to antigen group A, C, D, E, F, and had poor antigen cross-reactivity. It was further verified that the H9N2 avian influenza virus isolated in 2015 forms a new antigen group G.

TABLE 3-1. results of the micro-neutralization test for detecting antigenic changes of H9N2 avian influenza virus

Note: < indicates that the antibody titer was less than 10.

TABLE 3-2 results of the micro-neutralization test for detecting antigenic changes of H9N2 avian influenza virus

Note: < indicates that the antibody titer was less than 10.

Combining the experimental results, A/Chicken/Beijing/0701/2015 (i.e. BJ/15 strain) is selected for subsequent experiments.

Example 2 preparation of recombinant herpesvirus of turkeys expressing H9N2 subtype avian influenza Virus H9 protein (i.e., recombinant Virus rHVT-H9)

Construction of recombinant plasmid pBAC-GFP-US

1. The nucleotide sequence of the GFP gene (NCBI SEQ ID NO: LC336974.1) was artificially synthesized and inserted into a pUC19 vector to construct a plasmid pUC-GFP. The plasmid pUC-GFP was used as a template, and a primer pair consisting of GFP-F and GFP-R was used for PCR amplification to obtain a DNA fragment of about 720 bp.

2. And (3) taking the DNA fragment obtained in the step (1), carrying out double enzyme digestion by using restriction enzymes NheI and XbaI, and recovering the enzyme digestion fragment.

3. The pcDNA3.1+ vector was digested with restriction enzymes NheI and XbaI, and the vector backbone of about 5.4kb was recovered.

4. And (3) connecting the enzyme digestion fragment obtained in the step (2) with the vector framework obtained in the step (3) to obtain the recombinant plasmid pcDNA-GFP.

5. And (3) carrying out PCR amplification by using the recombinant plasmid pcDNA-GFP obtained in the step (4) as a template and adopting a primer pair consisting of GFP exp-F and GFP exp-R to obtain a DNA fragment of about 1668 bp. The DNA fragment is a GFP eukaryotic expression cassette containing a CMV promoter and a bGH terminator.

6. And (5) carrying out double enzyme digestion on the DNA fragment obtained in the step (5) by using restriction enzymes BamHI and HindIII, and recovering the enzyme digestion fragment.

7. The vector backbone was recovered by double digestion with the restriction enzymes BamHI and HindIII from the pBeloBAC11 plasmid.

8. And (4) connecting the enzyme digestion fragment obtained in the step (6) with the vector framework obtained in the step (7) to obtain the recombinant plasmid pBAC-GFP.

9. Chicken embryo fibroblasts (CEF cells for short) were infected with HVT, and genomic DNA of HVT was extracted 5 days after infection.

10. Using the genomic DNA of HVT as a template, a primer pair consisting of left arm-F and left arm-R was used for PCR amplification to obtain a DNA fragment of about 2061bp (i.e., the left homology arm). Using genomic DNA of HVT as a template, PCR amplification was performed using a primer pair consisting of right arm-F and right arm-R to obtain a DNA fragment of about 2696bp (i.e., right homology arm).

11. The pUC19 plasmid was digested with restriction enzymes SalI and KpnI, and the vector backbone was recovered.

12. And (3) taking the left homologous arm obtained in the step (10), carrying out double enzyme digestion by using restriction enzymes SalI and BamHI, and recovering a digestion fragment 1. Taking the right homologous arm obtained in the step 10, carrying out double enzyme digestion by using restriction enzymes BamHI and KpnI, and recovering an enzyme digestion fragment 2.

13. And (3) connecting the enzyme-digested

fragments

1 and 2 obtained in the step (12) with the vector skeleton obtained in the step (11) to obtain a recombinant plasmid pUC-US.

14. The recombinant plasmid pBAC-GFP was digested with restriction enzyme BamHI, and the digested fragment of about 9139bp was recovered.

15. The recombinant plasmid pUC-US was digested with the restriction enzyme BamHI, and the vector backbone of about 7400bp was recovered.

16. And (3) connecting the enzyme digestion fragment obtained in the step (14) with the vector framework obtained in the step (15) to obtain the recombinant plasmid pBAC-GFP-US.

II, obtaining positive clone of recombinant virus HVT-BAC

1. Purified HVT genome DNA and recombinant plasmid pBAC-GFP-US are co-transfected into CEF cells, and through homologous recombination, recombinant virus HVT-BAC capable of emitting green fluorescence (green fluorescence gene GFP is used as a screening marker) is obtained.

2. After completion of step 1, the recombinant virus HVT-BAC was purified and enriched, respectively.

3. After step 2, genomic DNAs of the recombinant viruses HVT-BAC were extracted, respectively, and then transformed into E.coli DH10B competent cells using a BIO-RAD electrotransformation machine, respectively, and then plated on LB solid plates containing chloramphenicol (Cm) after 2h rejuvenation, and cultured overnight at 37 ℃ in an inverted manner.

The clone capable of growing on the plate is the recombinant virus HVT-BAC positive clone.

Thirdly, construction of recombinant plasmid pcDNA-pec-H9

1. A double-stranded DNA molecule of the pec promoter (NCBI SEQ ID NO: AF428265.1) was synthesized.

2. The double-stranded DNA molecule synthesized in step 1 was ligated with pUC57 vector to obtain recombinant plasmid pUC-pec.

3. And carrying out PCR amplification by using a primer pair consisting of pec-F and pec-R by using the recombinant plasmid pUC-pec as a template to obtain a DNA fragment of about 561 bp.

4. And (3) carrying out double enzyme digestion on the DNA fragment obtained in the step (3) by using restriction enzymes NheI and KpnI, and recovering the enzyme digestion fragment.

5. The pcDNA3.1+ vector was digested with restriction enzymes NheI and KpnI, and the vector backbone of about 5.4kb was recovered.

6. And (4) connecting the enzyme digestion fragment obtained in the step (4) with the vector skeleton obtained in the step (5) to obtain the recombinant plasmid pcDNA-pec. The recombinant plasmid pcDNA-pec contains the pec promoter and the bGH terminator.

7. The BJ/15 virus strain was inoculated into SPF chick embryos (for virus amplification), allantoic fluid was collected and RNA was extracted using Roche RNA extraction kit, and then the RNA was reverse transcribed into cDNA using MLV reverse transcription kit.

8. And (3) carrying out PCR amplification by using the cDNA obtained in the step (7) as a template and adopting a primer pair consisting of H9-F and H9-R to obtain a DNA fragment of about 1697 bp. The DNA fragment contains a nucleotide sequence (namely H9 gene) shown as a sequence 1 in a sequence table and encodes H9 protein shown as a sequence 2in the sequence table.

9. Taking the DNA fragment obtained in the step 8, carrying out double enzyme digestion by using restriction enzymes KpnI and BamHI, and recovering an enzyme digestion fragment.

10. Taking the recombinant plasmid pcDNA-pec, carrying out double enzyme digestion by using restriction enzymes KpnI and BamHI, and recovering a vector framework.

11. And (3) connecting the enzyme digestion fragment obtained in the step (9) with the vector skeleton obtained in the step (10) to obtain a recombinant plasmid pcDNA-pec-H9.

The recombinant plasmid pcDNA-pec-H9 contains an H9 expression cassette. The H9 expression cassette includes the pec promoter, the H9 gene, and the bGH terminator. The pec promoter comprises the CMV enhancer and the chicken β -actin promoter.

The nucleotide sequence of the H9 expression cassette is shown as sequence 3 in the sequence table. From the 5' end of the sequence 3 in the sequence table, the 1 st to 274 th positions are CMV enhancers, the 275 th to 561 th positions are chicken beta-actin promoters, the 568 th to 2250 th positions are H9 genes, and the 2350 th to 2573 th positions are bGH terminators. The 1 st to 561 th positions of the sequence 3 in the sequence table from the 5' end are pec promoters.

Fourthly, preparation of recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein

SW102 genetically engineered bacteria and peGFP-galk are described in the following documents: norm S, costatino N, Coort D L, Jenkins N A, and Copeland N G.simple and highlyy impact BAC regringbinding using galK selection.2005; 33: e36-e36.

1. And (3) introducing the HVT-BAC positive clone into the SW102 genetic engineering bacteria to obtain a recombinant bacterium, and then preparing the electric transformation competence of the recombinant bacterium.

2. PCR amplification is carried out by taking pepGFP-galk as a template and adopting a primer pair consisting of homo galk-F (containing 50bp homology arm) and homo galk-R (containing 50bp homology arm) to obtain a DNA fragment of about 1331 bp.

3. And (3) taking the DNA fragment obtained in the step (2), purifying, then electrically transforming to the electric transformation competence of the recombinant bacteria prepared in the step (1) for recombination, then coating the recombinant bacteria on an LB solid plate containing galactose, and carrying out inverted culture at 32 ℃ for overnight.

The clone capable of growing on the plate is SW102 strain containing recombinant plasmid HVT-BAC-Galk. In contrast to the HVT-BAC positive clone, the recombinant plasmid HVT-BAC-Galk inserts the Galk sequence containing em7 promoter only after 95322 nt.

4. The recombinant plasmid pcDNA-pec-H9 is used as a template, and a primer pair consisting of homo H9-F (containing 50bp homology arm) and homo H9-R (containing 50bp homology arm) is adopted for PCR amplification to obtain a DNA fragment of about 2673 bp.

5. The DNA fragment obtained in step 4 was taken, purified, and then electrically transformed into a recombinant plasmid HVT-BAC-Galk-containing SW102 strain, which was competent for recombination, and then spread on an LB solid plate containing 2-deoxy-galactose (DOG), and cultured overnight at 32 ℃ in an inverted state.

The clone capable of growing on the plate is SW102 strain containing recombinant plasmid HVT-BAC-H9. In contrast to the recombinant plasmid HVT-BAC-Galk, the recombinant plasmid HVT-BAC-H9 replaced only the em 7-initiated Galk sequence with the H9 eukaryotic expression cassette.

The schematic diagrams of steps 1 to 5 above are shown in detail in fig. 3.

6. Construction of pX458-sgRNA vector

(1) And (5) designing and artificially synthesizing sgRNA-F and sgRNA-R by taking BAC sequences outside the GFP eukaryotic expression cassette in the step one as target sites.

(2) And (4) preparing an annealing reaction system. The annealing reaction system was 10. mu.L, consisting of 1. mu.L of an aqueous solution of sgRNA-F (concentration: 100nM), 1. mu.L of an aqueous solution of sgRNA-R (concentration: 100nM), 1. mu.L of 10 XT 4ligase Buffer, 1. mu.LT 4PNK, and 6. mu.L ddH₂And (C) O.

(3) And (3) after the step (2) is finished, taking the annealing reaction system, and carrying out annealing reaction to form a DNA molecule I.

And (3) annealing procedure: 30min at 37 ℃ and 5min at 95 ℃, and the temperature of PCR is reduced to 25 ℃ in a gradient manner at a speed of 0.1 ℃ per second, 5min at 25 ℃ and 5min at 4 ℃.

(4) A pX458 vector (a product of Addgene company, the product catalog number is 48138; the pX458 vector also comprises a Cas9 gene) is taken and cut by restriction enzyme BbsI, and a vector framework is recovered.

(5) And (4) connecting the DNA molecule I obtained in the step (3) with the vector framework obtained in the step (4) to obtain a pX458-sgRNA vector.

The pX458-sgRNA vector was sequenced. Based on the sequencing results, the pX458-sgRNA vector was structurally described as follows: the recognition site of the restriction enzyme BbsI of the pX458 vector was inserted with the DNA molecule I. The nucleotide sequence of the DNA molecule I is 5'-CACCGAATGCGGATCTCTACGATAAGTTT-3'.

7. The plasmid of SW102 strain containing the recombinant plasmid HVT-BAC-H9 was extracted to obtain the recombinant plasmid HVT-BAC-H9.

8. The recombinant plasmid HVT-BAC-H9, the recombinant plasmid pX458-sgRNA and the homologous sequence donor gene of HVT US region (with the genomic DNA of HVT as a template, a primer pair consisting of donor-F and donor-R is adopted for PCR amplification to obtain a DNA fragment (139462 nt-141036nt of the genomic DNA of HVT)) are jointly transfected into CEF cells, and the BAC sequence is replaced by the donor gene through homologous recombination induced by CRIPSR/Cas9, so that the recombinant herpesvirus of turkeys (namely recombinant virus rHVT-H9) which are only inserted into an H9 expression cassette and express H9N2 subtype avian influenza virus H9 protein are obtained. Since the rHVT-H9 does not contain GFP, the rHVT-H9 could be successfully screened by screening plaques that do not contain green fluorescence.

The schematic diagrams of steps 6 to 8 described above are shown in detail in fig. 4.

Thus, the recombinant virus rHVT-H9 was obtained by modifying the HVT genome as follows: an H9 expression cassette was inserted between 95322nt and 95323nt of the genomic UL region of HVT. The H9 expression cassette includes the pec promoter, the H9 gene, and the bGH terminator.

The recombinant virus rHVT-CMV-H9 was constructed according to the above procedure. The recombinant virus rHVT-CMV-H9 is obtained by modifying HVT genome as follows: the CMV-H9 expression cassette was inserted between 95322nt and 95323nt of the UL region of the HVT genome. The CMV-H9 expression cassette differs from the H9 expression cassette only in that the pec promoter (shown in the sequence listing at positions 1 to 561 from the 5' end of sequence 3) is replaced with a CMV promoter. The nucleotide sequence of the CMV promoter is shown as a sequence 4 in a sequence table.

Example 3 identification of recombinant Virus rHVT-H9

First, gene level identification

Extracting the genome DNA of the recombinant virus rHVT-H9, taking the genome DNA as a template, and carrying out PCR amplification by adopting a primer pair consisting of H9-F and H9-R to obtain a PCR amplification product.

As a result, the PCR amplification product contained a DNA fragment of about 1683 bp. It can be seen that the H9 gene has been completely inserted into the HVT genome.

Two, indirect immunofluorescence assay

1. A24-well cell culture plate was taken, and 500. mu.L of a culture medium (about 2.25X 10) containing CEF cells was added thereto⁵CEF cells/well), 37 ℃ 5% CO₂Culturing until the CEF cells grow into a monolayer.

2. After completion of step 1, the recombinant virus rHVT-H9 (inoculum size 100 PFU/well) was inoculated at 37 ℃ with 5% CO₂And culturing for 72 h.

3. After step 2 was completed, the medium was discarded, washed 1 time with PBS buffer (pH 7.2, 0.01 mM), and 500. mu.L of a pre-cooled fixative (consisting of 3 parts by volume of ethanol and 2 parts by volume of acetone) was added to each well and fixed for 20 min.

4. After step 3, discarding the liquid phase, washing with PBS buffer solution of 0.01mM and pH7.2 for 5min for 3 times; then, 300. mu.L of a dilution of a mouse polyclonal antibody (prepared by mixing 1 part by volume of the mouse polyclonal antibody with 49 parts by volume of PBS buffer solution of pH7.2 and 0.01 mM; the mouse polyclonal antibody is prepared by immunizing a mouse with the BJ/15 virus strain by nasal cavity inoculation, immunizing for 1 time every two weeks, immunizing for 3 times in total, then killing and collecting serum), and incubating overnight at 4 ℃.

5. After step 4, discarding the liquid phase, washing with PBS buffer solution (pH7.2, 0.01 mM) for 3 times (5 min each time); then, a green Fluorescent (FITC) -labeled anti-mouse secondary antibody was added for IFA staining and observed under an inverted fluorescence microscope.

Recombinant virus rHVT-H9 was replaced with HVT according to the above procedure, and all other procedures were unchanged as a control.

The results of the experiment are shown in FIG. 5. The result shows that the recombinant virus rHVT-H9 can generate obvious cytopathy after infecting CEF cells for 72H, form plaque which can be specifically bound by the polyclonal antibody of the mouse anti-H9 and generate green fluorescence after IFA staining, and the HVT control can form plaque which cannot be specifically bound by the polyclonal antibody of the mouse anti-H9.

Third, Western blot identification

1. A6-well cell culture plate was prepared, and 2nL of a cell culture medium (about 1X 10) containing CEF cells was added thereto⁶CEF cells/well), 37 ℃ 5% CO₂Culturing until the CEF cells grow into a monolayer.

2. After completion of step 1, the recombinant virus rHVT-H9 (inoculation dose 500 PFU/well) was inoculated at 37 ℃ with 5% CO₂And culturing for 72 h.

3. The cells that completed step 2 were collected by digesting with trypsin, and then centrifuging.

4. And 3, after the step 3 is finished, taking the cells, and cracking the cells to obtain cell lysate.

5. After step 4, the cell lysate was subjected to SDS-PAGE and Western blot (using a mouse polyclonal antibody (the mouse polyclonal antibody of step 4 in the same step) as the primary antibody, a Horse Radish Peroxidase (HRP) -labeled goat anti-mouse antibody as the secondary antibody to detect H9 protein, and β -actin protein as the internal reference).

The results of the experiment are shown in FIG. 6. The results show that the lysate of CEF cells infected by the recombinant virus rHVT-H9 can detect a band specific to H9 at the size position of 78KD, but the CEF cells infected by HVT do not have the specific band.

Example 4 immunoprotection evaluation of recombinant Virus rHVT-H9 against AIV subtype H9N2

1. 30 SPF chickens of 1 day old were randomly divided into three groups of 10 recombinant virus rHVT-H9, rHVT-CMV-H9 and control. Then the following treatment is carried out:

recombinant virus rHVT-H9 group: on the 1 st day of the experiment, the neck was inoculated subcutaneously with recombinant virus rHVT-H9 at an inoculation dose of 3000 PFU/mouse; on day 35 of the experiment, the nasal cavity was inoculated with BJ/15 strain at a dose of 10⁴ELD₅₀；

Recombinant virus rHVT-CMV-H9 group: on the 1 st day of the experiment, the neck was inoculated subcutaneously with recombinant virus rHVT-CMV-H9 at an inoculation dose of 3000 PFU/mouse; on day 35 of the experiment, the nasal cavity was inoculated with BJ/15 strain at a dose of 10⁴ELD₅₀；

Control group: on the 1 st day of the experiment, the neck was inoculated with HVT subcutaneously at an inoculation dose of 3000 PFU/mouse; on day 35 of the experiment, the nasal cavity was inoculated with BJ/15 strain at a dose of 10⁴ELD₅₀。

2. After completing step 1, on the 38 th day (the 3 rd day) and the 40 th day (the 5 th day) of the experiment, oral cavity and cloaca swabs were collected from each chicken to determine whether to expel toxin.

The results are shown in Table 4. The results show that the chickens in the control group expel toxin through oral cavity and cloaca, while the chickens in the recombinant virus rHVT-H9 group and the recombinant virus rHVT-CMV-H9 group do not expel toxin.

TABLE 4 toxin expelling test after challenge of immunized chickens

Note: a, mean. + -. standard deviation in EID₅₀/mL。

3. After completion of step 1, on the 38 th (day 3) and 40 th (day 5) of the experiment, the internal organs, trachea, lung, spleen and kidney were collected from each chicken, and the viral titer of the internal organs was titrated to detect the replication of the BJ/15 virus strain.

The results are shown in Table 5. The results show that the BJ/15 strain is able to replicate efficiently in the chickens of the control group; the chickens inoculated with the recombinant virus rHVT-H9 and the chickens inoculated with the recombinant virus rHVT-CMV-H9 can completely prevent the BJ/15 virus strain from replicating in organs.

TABLE 5 toxin expelling test after challenge of immunized chickens

Note: a, mean. + -. standard deviation in EID₅₀/mL。

4. Venous blood was collected and serum was isolated from each of experiment days 8, 15, 22, 29 and 36, and then the titer of serum antibodies was measured using the Hemagglutination Inhibition (HI) method (described in Cox N, Webster R G, Krauss S, Guan Y, Hay A, Yu K, et al, WHO Manual on antibacterial Influenza and Surveillance,2nd edition.2005) and the Virus Neutralization (VN) method (described in Cottey R, Rowe C A, and Bender B S. Influenza Virus. Current Protocols in Immunology; 2001: t 19.11).

The results of the serum HI antibody titer test are shown in FIG. 7(rHVT-pec-H9 is recombinant virus rHVT-H9, and rHVT-CMV-H9 is recombinant virus rHVT-CMV-H9). The result shows that the serum HI antibody can be detected 2 weeks after the recombinant virus rHVT-H9 is immunized, and the HI antibody effect level is continuously improved along with the time extension; 4-5 weeks after immunization, HI antibody induced by recombinant virus rHVT-H9 was slightly higher than recombinant virus rHVT-CMV-H9; the geometric mean titers of HI antibodies induced 5 weeks after immunization by recombinant virus rHVT-H9 and recombinant virus rHVT-CMV-H9 were 38.4 and 25.6, respectively.

The result of the serum VN antibody titer test is shown in FIG. 8(rHVT-pec-H9 is recombinant virus rHVT-H9, and rHVT-CMV-H9 is recombinant virus rHVT-CMV-H9). The results show that the same serum VN antibodies were also able to detect H9 specific antibodies two weeks after immunization; at 5 weeks after immunization, VN antibodies induced by the recombinant virus rHVT-H9 were slightly higher than VN antibodies induced by the recombinant virus rHVT-CMV-H9, the recombinant virus rHVT-H9 and the recombinant virus rHVT-CMV-H9 with geometric mean titers of 120 and 92, respectively.

The results show that the recombinant virus rHVT-H9 has excellent immune protection effect on H9N2 subtype AIV.

<110> university of agriculture in China

<120> recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1683

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 1

atggagacag tatcactaat aactatacta ctagtagcag cagtaagcaa tgcagataaa 60

atctgcatcg gctatcaatc aacaaactcc acagaaactg tggacacact aacagaaaac 120

aatgtccctg tgacacatgc caaagaactg ctccacacag agcataatgg gatgctgtgt 180

gcaacaagct tgggacaacc tcttatttta gacacctgca ccattgaagg gctaatctat 240

ggcaatcctt attgtgatct atcgctggaa ggaagagaat ggtcctatat agtcgagaga 300

ccatcagctg ttaacggatt gtgttacccc gggaatgtag aaaatctaga agagttaagg 360

tcacttttta gttctgctag gtcttatcaa agaatccaga ttttcccaga cacaatctgg 420

aatgtatctt acgatgggac aagcgcagca tgctcaggtt cattctacaa aagcatgaga 480

tggttgactc aaaagaacgg cgattaccct atccaagacg cccaatacac aaataatcaa 540

gggaagaaca ttcttttcat gtggggcata aatcaaccac ccaccgatac tacgcagaga 600

aatctgtaca cgagaaccga cacaacaacg agtgtggcaa cagaagaaat aaatagggtc 660

ttcaaaccat tgataggacc aaggcctctt gtcaacggtt tgatgggaag aattgattat 720

tattggtcgg tattgaaacc gggtcaaaca ctgcgaataa aatctgatgg gaatctaata 780

gccccatggt ttggacacat tctttcagga gagagccacg gaagaattct gaagactgat 840

ttaaaaaggg gtagctgcac agtgcaatgt cagacagaga aaggtggctt gaacacaaca 900

ttgccattcc aaaacataag taagtatgca tttggaaact gctcaaaata cattggcata 960

aagagtctca aacttgcagt tggtctgagg aatgtgcctt ctagatctag tagaggacta 1020

tttggggcca tagcagggtt tatagaggga ggttggtcag gactagttgc tggttggtat 1080

gggttccagc attcaaatga ccaaggagtt ggtatggcag cagatagaga ctcaacccaa 1140

aaggcaattg ataaaataac atccaaagtg aataatatag tcgacaaaat gaacaagcag 1200

tatgaaatca ttgatcatga attcagtgag gtagaaacta gacttaacat gatcaataat 1260

aagattgatg atcaaatcca ggatatatgg gcatataatg cagaattgct agttctgctt 1320

gaaaaccaga aaacactcga tgagcacgac gcaaatgtaa acaatctata taataaagta 1380

aagagggcgt tgggttccaa tgcggtggaa gatggaaaag gatgtttcga gctataccac 1440

aaatgtgatg accaatgcat ggagacaatt cggaatggga cctacaacag aaggaagtat 1500

cgagaggagt caaaattaga aagacagaaa atagaggggg tcaagctgga atctgaagga 1560

acttacaaaa tcctcaccat ttattcgact gtcgcctcat ctcttgtgat tgcaatgggg 1620

tttgctgcct tcttgttctg ggccatgtcc aatgggtctt gcagatgcaa catttgtata 1680

tga 1683

<210> 2

<211> 560

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 2

Met Glu Thr Val Ser Leu Ile Thr Ile Leu Leu Val Ala Ala Val Ser

1 5 10 15

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

20 25 30

Thr Val Asp Thr Leu Thr Glu Asn Asn Val Pro Val Thr His Ala Lys

35 40 45

Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Ser Leu

50 55 60

Gly Gln Pro Leu Ile Leu Asp Thr Cys Thr Ile Glu Gly Leu Ile Tyr

65 70 75 80

Gly Asn Pro Tyr Cys Asp Leu Ser Leu Glu Gly Arg Glu Trp Ser Tyr

85 90 95

Ile Val Glu Arg Pro Ser Ala Val Asn Gly Leu Cys Tyr Pro Gly Asn

100 105 110

Val Glu Asn Leu Glu Glu Leu Arg Ser Leu Phe Ser Ser Ala Arg Ser

115 120 125

Tyr Gln Arg Ile Gln Ile Phe Pro Asp Thr Ile Trp Asn Val Ser Tyr

130 135 140

Asp Gly Thr Ser Ala Ala Cys Ser Gly Ser Phe Tyr Lys Ser Met Arg

145 150 155 160

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

165 170 175

Thr Asn Asn Gln Gly Lys Asn Ile Leu Phe Met Trp Gly Ile Asn Gln

180 185 190

Pro Pro Thr Asp Thr Thr Gln Arg Asn Leu Tyr Thr Arg Thr Asp Thr

195 200 205

Thr Thr Ser Val Ala Thr Glu Glu Ile Asn Arg Val Phe Lys Pro Leu

210 215 220

Ile Gly Pro Arg Pro Leu Val Asn Gly Leu Met Gly Arg Ile Asp Tyr

225 230 235 240

Tyr Trp Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Ile Lys Ser Asp

245 250 255

Gly Asn Leu Ile Ala Pro Trp Phe Gly His Ile Leu Ser Gly Glu Ser

260 265 270

His Gly Arg Ile Leu Lys Thr Asp Leu Lys Arg Gly Ser Cys Thr Val

275 280 285

Gln Cys Gln Thr Glu Lys Gly Gly Leu Asn Thr Thr Leu Pro Phe Gln

290 295 300

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

305 310 315 320

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

325 330 335

Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp

340 345 350

Ser Gly Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln

355 360 365

Gly Val Gly Met Ala Ala Asp Arg Asp Ser Thr Gln Lys Ala Ile Asp

370 375 380

Lys Ile Thr Ser Lys Val Asn Asn Ile Val Asp Lys Met Asn Lys Gln

385 390 395 400

Tyr Glu Ile Ile Asp His Glu Phe Ser Glu Val Glu Thr Arg Leu Asn

405 410 415

Met Ile Asn Asn Lys Ile Asp Asp Gln Ile Gln Asp Ile Trp Ala Tyr

420 425 430

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

435 440 445

His Asp Ala Asn Val Asn Asn Leu Tyr Asn Lys Val Lys Arg Ala Leu

450 455 460

Gly Ser Asn Ala Val Glu Asp Gly Lys Gly Cys Phe Glu Leu Tyr His

465 470 475 480

Lys Cys Asp Asp Gln Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asn

485 490 495

Arg Arg Lys Tyr Arg Glu Glu Ser Lys Leu Glu Arg Gln Lys Ile Glu

500 505 510

Gly Val Lys Leu Glu Ser Glu Gly Thr Tyr Lys Ile Leu Thr Ile Tyr

515 520 525

Ser Thr Val Ala Ser Ser Leu Val Ile Ala Met Gly Phe Ala Ala Phe

530 535 540

Leu Phe Trp Ala Met Ser Asn Gly Ser Cys Arg Cys Asn Ile Cys Ile

545 550 555 560

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<211> 2573

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 3

agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc 60

gttacataac ttacggtaaa tggcccgccg gctgaccgcc caacgacccc cgcccattga 120

cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 180

gggtggagta tttacggtaa actgcccatt ggcagtacat caagtgtatc atatgccaag 240

tacgccccct attgacgtca atgacggtaa atggatgcag tattttgtgc agcgatgggg 300

gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 360

gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 420

ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 480

tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct 540

ctgactgacc gcgtctagag gggtaccatg gagacagtat cactaataac tatactacta 600

gtagcagcag taagcaatgc agataaaatc tgcatcggct atcaatcaac aaactccaca 660

gaaactgtgg acacactaac agaaaacaat gtccctgtga cacatgccaa agaactgctc 720

cacacagagc ataatgggat gctgtgtgca acaagcttgg gacaacctct tattttagac 780

acctgcacca ttgaagggct aatctatggc aatccttatt gtgatctatc gctggaagga 840

agagaatggt cctatatagt cgagagacca tcagctgtta acggattgtg ttaccccggg 900

aatgtagaaa atctagaaga gttaaggtca ctttttagtt ctgctaggtc ttatcaaaga 960

atccagattt tcccagacac aatctggaat gtatcttacg atgggacaag cgcagcatgc 1020

tcaggttcat tctacaaaag catgagatgg ttgactcaaa agaacggcga ttaccctatc 1080

caagacgccc aatacacaaa taatcaaggg aagaacattc ttttcatgtg gggcataaat 1140

caaccaccca ccgatactac gcagagaaat ctgtacacga gaaccgacac aacaacgagt 1200

gtggcaacag aagaaataaa tagggtcttc aaaccattga taggaccaag gcctcttgtc 1260

aacggtttga tgggaagaat tgattattat tggtcggtat tgaaaccggg tcaaacactg 1320

cgaataaaat ctgatgggaa tctaatagcc ccatggtttg gacacattct ttcaggagag 1380

agccacggaa gaattctgaa gactgattta aaaaggggta gctgcacagt gcaatgtcag 1440

acagagaaag gtggcttgaa cacaacattg ccattccaaa acataagtaa gtatgcattt 1500

ggaaactgct caaaatacat tggcataaag agtctcaaac ttgcagttgg tctgaggaat 1560

gtgccttcta gatctagtag aggactattt ggggccatag cagggtttat agagggaggt 1620

tggtcaggac tagttgctgg ttggtatggg ttccagcatt caaatgacca aggagttggt 1680

atggcagcag atagagactc aacccaaaag gcaattgata aaataacatc caaagtgaat 1740

aatatagtcg acaaaatgaa caagcagtat gaaatcattg atcatgaatt cagtgaggta 1800

gaaactagac ttaacatgat caataataag attgatgatc aaatccagga tatatgggca 1860

tataatgcag aattgctagt tctgcttgaa aaccagaaaa cactcgatga gcacgacgca 1920

aatgtaaaca atctatataa taaagtaaag agggcgttgg gttccaatgc ggtggaagat 1980

ggaaaaggat gtttcgagct ataccacaaa tgtgatgacc aatgcatgga gacaattcgg 2040

aatgggacct acaacagaag gaagtatcga gaggagtcaa aattagaaag acagaaaata 2100

gagggggtca agctggaatc tgaaggaact tacaaaatcc tcaccattta ttcgactgtc 2160

gcctcatctc ttgtgattgc aatggggttt gctgccttct tgttctgggc catgtccaat 2220

gggtcttgca gatgcaacat ttgtatatga ggatccacta gtccagtgtg gtggaattct 2280

gcagatatcc agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat 2340

cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 2400

ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 2460

cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 2520

gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atg 2573

<210> 4

<211> 685

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 4

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660

ctggctagcg tttaaactta agctt 685

Claims

1. A recombinant turkey herpesvirus, comprising: the recombinant herpesvirus of turkeys is obtained by modifying the genome of the original herpesvirus of turkeys as follows: the DNA molecule of the coding gene containing H9N2 subtype avian influenza virus H9 protein is increased;

the H9N2 subtype avian influenza virus H9 protein is a1) or a2) as follows:

a2) and (b) fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2in the sequence table.

2. The recombinant turkey herpesvirus as in claim 1, wherein: the encoding gene of the H9N2 subtype avian influenza virus H9 protein is a DNA molecule shown in a sequence 1 of a sequence table.

3. The recombinant turkey herpesvirus as in claim 1, wherein: the transformation is realized in the following manner: inserting specific DNA molecules between 95322nt and 95323nt of the UL region of the genome of the herpes virus of the starting turkey; the specific DNA molecule contains a promoter, a coding gene of the H9N2 subtype avian influenza virus H9 protein and a terminator.

4. The recombinant turkey herpesvirus as in claim 3, wherein: the specific DNA molecule also contains an enhancer; the enhancer is located upstream of the promoter.

5. The recombinant turkey herpesvirus as in claim 4, wherein: the specific DNA molecule sequentially contains the enhancer, the promoter, the coding gene of the H9N2 subtype avian influenza virus H9 protein and the terminator from upstream to downstream.

6. The specific DNA molecule contains a promoter, a coding gene of H9N2 subtype avian influenza virus H9 protein and a terminator;

the H9N2 subtype avian influenza virus H9 protein is a1) or a2) as follows:

7. The specific DNA molecule of claim 6, wherein: the specific DNA molecule also contains an enhancer; the enhancer is located upstream of the promoter.

8. The specific DNA molecule of claim 7, wherein: the specific DNA molecule sequentially contains the enhancer, the promoter, the coding gene of the H9N2 subtype avian influenza virus H9 protein and the terminator from upstream to downstream.

The H9N2 subtype avian influenza virus H9 protein is a1) or a2) as follows:

10. The gene encoding H9 protein of avian influenza virus subtype H9N2 as claimed in claim 9.

11. Use of a recombinant herpesvirus of turkeys as claimed in any one of claims 1 to 5, in the manufacture of a vaccine for the prevention of avian influenza virus.

12. Use of the H9N2 subtype avian influenza virus H9 protein of claim 9 in the manufacture of a vaccine for the prophylaxis of avian influenza virus.

13. Use of the gene encoding H9 protein of H9N2 subtype avian influenza virus according to claim 9 in the preparation of a vaccine for the prophylaxis of avian influenza virus.

14. Use of a specific DNA molecule according to any one of claims 6 to 8 for the preparation of a vaccine for the prophylaxis of avian influenza virus.

15. A vaccine for the prophylaxis of avian influenza virus, which comprises the recombinant herpesvirus of turkeys as claimed in any one of claims 1 to 5.