ILTV gD protein antigen epitope expression cassette, recombinant virus and application

Abstract

The invention discloses an ILTV gD protein antigen epitope expression cassette, a recombinant virus and application, and relates to the field of gene and protein engineering. The ILTV gD protein antigen epitope expression cassette provided by the invention comprises T and B cell epitopes of gD protein derived from an infectious laryngotracheitis virus WG strain. The recombinant virus rH120-gD-T / B is obtained by replacing a 5ab gene of an infectious bronchitis virus H120 strain with a gene for coding an ILTV gD protein antigen epitope expression cassette. The recombinant virus has good genetic stability, can be used for developing a safe and effective IB-ILT bivalent live vector vaccine, and solves the problem of poor stability of the current recombinant IBV.

Description

Technical Field

[0001] The present invention relates to the technical field of gene and protein engineering, and particularly relates to an ILTV gD protein antigenic epitope expression cassette, a recombinant virus and applications thereof. Background Art

[0002] Infectious bronchitis (IB) in chickens is an acute and highly contagious avian disease, and its pathogen is Infectious bronchitis virus (IBV). IBV belongs to the order Nidovirales, the family Coronaviridae, the genus Gammacoronavirus, and is an enveloped virus with a round or irregular morphology. The genome of IBV is a single-stranded positive-sense RNA that is not segmented, about 27.6 kb. Its genome has a cap structure at the 5' end and Poly(A) at the 3' end. Sequentially from the 5'-3' end, it is: 5'UTR-la / 1ab-S-3a-3b-E-M-5a-5b-N-3'UTR. At present, due to its convenient immunization method and effectiveness, live attenuated IBV vaccines are widely used for the prevention and control of IB. The H120 strain is the most widely used IBV vaccine strain globally in the past 60 years. The H120 strain belongs to the Mass serotype and is a live attenuated vaccine obtained by serial passage and attenuation in chicken embryos. Its safety and effectiveness have been widely verified.

[0003] With the in-depth study of the unique transcription mechanism of coronaviruses and the development of reverse genetic systems, it has become possible to develop vaccines that express foreign genes using coronaviruses as vectors. Current research has found that recombinant IBV is usually genetically unstable, but after foreign genes replace non-essential accessory genes, the stability of recombinant viruses is usually maximized. The stable expression of foreign proteins in recombinant IBV may depend on various factors, including the genetic backgrounds of the vector virus and foreign genes, the insertion sites of foreign genes, and the importance of the replaced viral genes. Some studies have shown that the Renilla luciferase gene (hRluc) was used to replace the ORF3, ORF5, and the intergenic region IR of the IBV genome respectively. The results showed that after the hRluc gene replaced ORF5, the stability of recombinant IBV was the highest, but the hRluc gene could only be stably inherited up to 12 generations when detected at the nucleic acid level.

[0004] With the continuous development of molecular biology, there has been a more in-depth study of the genomic structure and protein function of pathogens. Vaccines based on antigenic epitopes have begun to show good application prospects. An antigenic epitope is a specific region on an antigen molecule that can specifically bind to an antibody or a T cell receptor. These regions are usually a small part of the antigen molecule and can induce a specific immune response in the immune system, which is a key factor in vaccine design and immunotherapy. According to the cells that the antigen receptor binds to, antigenic epitopes can be divided into T cell epitopes and B cell epitopes. A T cell epitope is an epitope on an antigen peptide processed by an antigen-presenting cell (APC), usually a linear epitope composed of 8-12 amino acids, or it can also be a longer polypeptide chain. The recognition of T cell epitopes requires the participation of MHC molecules. Among them, CD4+ T cells recognize epitopes on MHC II molecules, and CD8+ T cells recognize epitopes on MHC I molecules. A B cell epitope is an epitope on an intact antigen molecule that has not been processed. Its recognition does not require the participation of MHC molecules, but rather exerts an antiviral effect by binding to the B cell receptor (BCR) to activate B lymphocytes to secrete antibodies. Current research has found that truncating the length of foreign genes can usually improve the stability of recombinant IBV. Selecting protective antigenic epitopes and using IBV as a vector backbone to construct recombinant IBV expressing multiple epitopes is an effective method to solve the problem of poor stability of current recombinant IBV.

[0005] Infectious laryngotracheitis (ILT) is an acute and highly contagious upper respiratory tract disease caused by Infectious laryngotracheitis virus (ILTV). The Office International des Epizooties (OIE) included it in Category B infectious diseases in 1999. Currently, attenuated vaccines are mainly used for the prevention and control of ILT at home and abroad. Although such vaccines can prevent clinical diseases, the reaction of animals after vaccination is relatively large, and latent infection leading to reversion of virulence is one of the important factors for the outbreak of ILT. Therefore, the prevention and control of ILT has become a difficult problem in the global poultry industry.

[0006] Current research on ILTV antigenic epitopes mainly focuses on the gB protein, and there are relatively few studies and reports on the antigenic epitopes of the gD protein. This invention focuses on studying the T-cell and B-cell epitopes of the gD protein of the ILTV WG strain for the development of a multi-epitope ILTV vaccine. The gD glycoprotein exists on the surface of virus particles and is the receptor for the binding of the virus to susceptible cells, playing a key role in virus adsorption, virus-host membrane fusion, and inducing a protective immune response in the body. In other herpesviruses, studies have confirmed that the BHV-1 gD protein can stimulate a stronger cellular immune response than the gB and gC proteins and induce the production of more efficient neutralizing antibodies; the antibody level induced by the HSV-1 gD protein is the highest and can completely resist the attack of a strong HSV strain; the subunit vaccine of the PRV gD protein can also resist the lethal attack of PRV. In the currently published research, there is a recombinant fowlpox virus (FPV) expressing the ILTV gD glycoprotein, but the interference of maternal antibodies is the main reason why the live recombinant fowlpox virus vaccine has not been widely promoted and applied. IBV is not affected by maternal antibodies and can be used for basic immunization at 1-day-old, shortening or eliminating the immune blank period of chicks, and is a good vaccine vector. In addition, although the genetically engineered subunit vaccine expressing the ILTV gD glycoprotein has good safety, its production is relatively complex, requires additional adjuvants and booster injections, and its production and immunization costs are relatively high, with limited application prospects in the field of poultry vaccines. Moreover, the antigenicity of the protein is affected by the selected expression system, and it is difficult to find a good expression system. Therefore, careful selection of the expression system is required when preparing the subunit vaccine. The IB-ILT combined live vector vaccine constructed in this invention has a low preparation cost and a convenient immunization method. It can be immunized by methods such as nasal and ocular drops, drinking water, and spraying, saving the clinical vaccine usage cost, and has high safety, solving the problems of large animal reactions after vaccination with the current ILT attenuated vaccine and the biosafety problems caused by latent infection. Summary of the Invention

[0007] In view of the above deficiencies in the prior art, this invention provides an ILTV gD protein antigenic epitope expression cassette, recombinant virus, and application thereof. The recombinant virus has good genetic stability and can provide effective protection against two diseases, IB and ILT, solving the problem of poor stability of the current recombinant IBV.

[0008] To achieve the above object, the technical solution adopted by this invention to solve its technical problems is: providing an ILTV gD protein antigenic epitope expression cassette, which includes at least one of the 4 ILTV antigenic epitope polypeptides.

[0009] Furthermore, the amino acid sequences of the 4 ILTV antigenic epitope polypeptides are respectively shown as SEQ ID N0.1 - 4.

[0010] Furthermore, the ILTV antigen epitope polypeptide sequences are connected by a flexible linker.

[0011] Furthermore, the amino acid sequence of the flexible linker is as shown in SEQ ID N0.5.

[0012] Furthermore, the order of the 4 ILTV antigen epitope polypeptides in the ILTV gD protein antigen epitope expression cassette is: FASQSTAAVTYDYILGRRALDALTIPAVGPYNRYLTRVSRGCDVVEL+GGG S+YRECGDVQLLSECAVQSAQMWALDYV+GGGS+GRFAQTALVTLEVND RCLKIGSRLNFLPSK+GGGS+PEDVEVPEDTEHDDPNSDPDYYNDMPAVIP VEETTKSSNAVSVPIFAAFVACAVALVGLLVWSIV.

[0013] Furthermore, the order of the 4 ILTV antigen epitope polypeptides in the ILTV gD protein antigen epitope expression cassette is: (P1)FASQSTAAVTYDYILGRRALDALTIPAVGPYNRYLTRVSRGCDV VEL+GGGS+(P2)YRECGDVQLLSECAVQSAQMWALDYV+GGGS+(P3)GRFAQTALVTLEVNDRCLKIGSRLNFLPSK+GGGS+(P4)PEDVEVPEDTE HDDPNSDPDYYNDMPAVIPVEETTKSSNAVSVPIFAAFVACAVALVGLLVW SIV.

[0014] Furthermore, the above 4 ILTV antigen epitope polypeptides are connected by GGGS (SEQ ID N0.5).

[0015] Furthermore, the ILTV antigen epitope polypeptide includes T cell epitopes and B cell epitopes derived from the gD protein of the chicken infectious laryngotracheitis virus WG strain.

[0016] Furthermore, the T cell epitope includes an amino acid sequence shown in at least one of SEQ ID N0.6 - 20.

[0017] Furthermore, the B cell epitope includes an amino acid sequence shown in at least one of SEQ ID N0.21 - 25.

[0018] A gene encoding the above ILTV antigen epitope polypeptide.

[0019] A recombinant virus, comprising a gene encoding the above-mentioned ILTV epitope polypeptide.

[0020] Furthermore, the recombinant virus uses the chicken infectious bronchitis virus H120 strain as a backbone, and replaces the 5ab gene of the chicken infectious bronchitis virus H120 strain with a gene encoding an ILTV gD protein epitope expression cassette.

[0021] Furthermore, the preparation method of the recombinant virus comprises the following steps: inserting the multi-epitope chimeric gene gD-T / B of the chicken infectious laryngotracheitis virus into the genome of the IBV H120 strain by the Red / ET homologous recombination technique, and rescuing the obtained recombinant strain rH120-gD-T / B.

[0022] A multi-epitope vaccine, comprising the above-mentioned recombinant virus.

[0023] The application of the above-mentioned ILTV gD protein epitope expression cassette, recombinant virus or multi-epitope vaccine in the preparation or as a drug for preventing chicken infectious bronchitis and / or chicken infectious laryngotracheitis.

[0024] In summary, the present invention has the following beneficial effects:

[0025] 1. The present invention uses the chicken infectious bronchitis virus H120 attenuated vaccine strain as a backbone, inserts the multi-epitope gene gD-T / B of the chicken infectious laryngotracheitis virus into the H120 genome, and the obtained recombinant virus rH120-gD-T / B has good genetic stability and can be stably inherited to the 30th generation, solving the problem of poor stability of the current recombinant IBV.

[0026] 2. The recombinant virus of the present invention can be used as a candidate vaccine strain for a bivalent vaccine for preventing chicken infectious bronchitis and chicken infectious laryngotracheitis, and has important practical application value.

[0027] 3. The vaccination method of the vaccine of the present invention is convenient, and can be vaccinated by methods such as nasal drip and eye drop, drinking water and spraying, saving the vaccine use cost in clinics, and having high safety, solving the problems of large animal reactions after vaccination with the current ILT attenuated vaccine and the biosafety problems caused by latent infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the construction of the infectious clone of the recombinant virus of the present invention;

[0029] Figure 2 It is a structural diagram of the epitope polypeptide protein of the ILTV gD protein epitope expression cassette;

[0030] Figure 3 It is an electrophoresis diagram of the restriction enzyme digestion identification of the intermediate vector pH120-Δ5ab-ccdB;

[0031] Figure 4 It is the electrophoresis diagram of the restriction enzyme digestion identification of the transcription vector pH120-gD-T / B;

[0032] Figure 5 It is the electrophoresis diagram of the RT-PCR identification of the recombinant virus rH120-gD-T / B;

[0033] Figure 6 It is the Western Blot identification result after the recombinant virus rH120-gD-T / B infects CK cells;

[0034] Figure 7 It is the proliferation curve diagram of the recombinant virus rH120-gD-T / B in chicken embryos;

[0035] Figure 8 It is the detection result diagram of the genetic stability of the foreign gene of the recombinant virus rH120-gD-T / B. Specific implementation mode

[0036] The principles and characteristics of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0037] In the present invention, it has been verified that the adjustment of the order of T cell and B cell epitopes will not affect the effect of the virus. Therefore, the order of T cell and B cell epitopes is not limited. That is, the adjustment of the order of T cell and B cell epitopes is also within the scope of this application, and those skilled in the art can adjust the order of T cell and B cell epitopes according to needs.

[0038] Example 1 Construction of recombinant IBV with chimeric ILTV gD protein antigen epitope expression cassette

[0039] The transcription vector pH120-gD-T / B is obtained by combining the Red / ET recombination engineering technology with the two-step strategy of ccdB counter-selection, and the ILTV gD protein antigen epitope expression cassette is chimerized into the transcription vector pH120, thereby obtaining a new transcription vector pH120-gD-T / B, as Figure 1 shown. The specific steps are as follows:

[0040] Among them, the above-mentioned ILTV gD protein antigen epitope expression cassette connects 4 ILTV antigen epitope polypeptides (SEQ ID N0.1 - 4) with a flexible linker (SEQ ID N0.5) at the same time. The polypeptide sequence is: (P1) FASQSTAAVTYDYILGRRALDALTIPAVGPYNRYLTRVSRGCDVVEL+GGGS+(P2) YRECGDVQLLSECAVQSAQMWALDYV+GGGS+(P3) GRFAQT ALVTLEVNDRCLKIGSRLNFLPSK+GGGS+(P4) PEDVEVPEDTEHDDPNSD PDYYNDMPAVIPVEETTKSSNAVSVPIFAAFVACAVALVGLLVWSIV.

[0041] The protein structures of the 4 ILTV antigen epitope polypeptides of the ILTV gD protein antigen epitope expression cassette are as Figure 2 shown.

[0042] Design primers Δ5ab-ccdB-F / Δ5ab-ccdB-R, as shown in Table 1. Using pBR322-ccdB-amp as a template, amplify the screening gene. The two ends of the amplified Δ5ab-ccdB gene fragment carry a homologous arm of the upstream and downstream of the H120 genomic 5ab gene. The PCR product was detected by 1% agarose gel electrophoresis. The target band was about 1400 bp, which was consistent with the expected size of 1421 bp. The PCR product was recovered and purified, the nucleic acid concentration was measured, and a small amount was sent to a sequencing company for sequence sequencing.

[0043] Table 1 Design of primers for amplifying the screening gene ccdB gene

[0044]

[0045] The purified screening gene fragment Δ5ab-ccdB and the pH120 vector were electrotransformed into an engineered bacterium expressing Redα / β for homologous recombination. After culturing on an LB plate for 16 h, single colonies were picked and identified by colony PCR. After the positive bacterial solution was amplified and cultured, the plasmid was extracted and identified by double digestion with XhoI and BstZ17I. The results are as Figure 3 shown.

[0046] It can be Figure 3 seen that the sizes of the enzyme digestion bands of the recombinant plasmid are exactly the same as the preset map, preliminarily indicating that the intermediate vector pH120-Δ5ab-ccdB is correctly constructed.

[0047] The correctly digested recombinant plasmid was sent to a sequencing company for sequencing. The results showed that all sequences were exactly the same as expected, indicating the successful construction of the intermediate plasmid pH120-Δ5ab-ccdB.

[0048] By the same method, the ILTV gD protein antigen epitope expression cassette was recombined with the intermediate vector pH120-Δ5ab-ccdB. The gD-T / B antigen epitope expression cassette replaced the screening gene ccdB. After the positive bacterial liquid was amplified and cultured by PCR identification, the plasmid was extracted. XhoI and BstZ17I double digestion identification was carried out, and the results were as Figure 4 shown.

[0049] As Figure 4 can be seen, the sizes of the enzyme digestion bands of the recombinant plasmid were exactly the same as the preset map, preliminarily indicating the correct construction of the recombinant plasmid pH120-gD-T / B. The correctly digested recombinant plasmid was sent to a sequencing company for sequencing. The results showed that all sequences were exactly the same as expected, indicating the successful construction of the transcription vector pH120-gD-T / B.

[0050] Example 2 Rescue and Identification of Recombinant Virus rH120-gD-T / B

[0051] (1) Virus rescue

[0052] Using the liposome transfection method, the constructed transcription vector pH120-gD-T / B and the helper plasmid pVAX-H120-N were co-transfected into BSR-T7 / 5 cells. 4 hours after transfection, the transfection solution was aspirated and discarded, and DMEM maintenance solution containing 2% FBS was added to continue culturing. 48 hours after transfection, the cells were harvested. After repeated freezing and thawing, the cell supernatant was inoculated into 9-11-day-old SPF chicken embryos. After incubating at 37°C for 48 hours, the allantoic fluid of the chicken embryos was harvested. After three passages of blind passage, the recombinant virus was identified.

[0053] (2) Recombinant virus identification

[0054] Take 200 μL of the virus solution and extract the virus RNA according to the instructions of the Axyprep Body Fluid Virus DNA / RNA Mini Extraction Kit. Using the above RNA as a template, the primer sequences and amplification lengths of the primers Δ5ab-JD-F / Δ5ab-JD-R are shown in Table 2. Prepare the reaction system according to the instructions of the one-step RT-PCR kit PrimeScript One Step RT-PCR Kit Ver.2. The reaction system is shown in Table 3. The amplification program: 50°C, 30 min; 94°C, 2 min; 94°C, 30 s, 56°C, 30 s, 72°C, 40 s, for a total of 32 cycles; 72°C, 5 min. The RT-PCR products were detected by 1% agarose gel electrophoresis and sent to a sequencing company for sequence sequencing. The results were as Figure 5 shown.

[0055] Table 2 Primer Design for Identification of gD-T / B Multi-epitope Gene

[0056]

[0057] Table 3 One-step RT-PCR Reaction System

[0058] Reaction components 25 μL reaction system Primescript 1Step Enzyme Mix 0.2 μL 2×1Step Buffer 12.5 μL Forward primer, 10 μM 1 μL Reverse primer, 10 μM 1 μL RNA 2 μL

[0059] It can be seen from Figure 5 that the target band (610 bp) is consistent with the expected size (3 bp of the start codon + 3 bp of the stop codon + 38 bp nucleotide sequence at the 5' end of the insertion site + 26 bp nucleotide sequence at the 3' end of the insertion site + 540 bp of the nucleotide sequences corresponding to 4 ILTV antigenic epitope polypeptides and 3 linker peptides of the ILTV gD protein antigenic epitope expression cassette), and the sequencing results show 100% consistency with the expected sequence, indicating the successful rescue of the recombinant virus rH120-gD-T / B.

[0060] The recombinant virus rH120-gD-T / B was inoculated into monolayer CK cells at an MOI of 0.1. After 48 h of infection, the culture medium was aspirated and discarded. After washing three times with PBS, a protein lysate containing 1% protease inhibitor was added, and the cells were lysed on ice for 20 min. The entire lysis mixture was transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000×g for 20 min at 4°C. 80 μL of the supernatant was aspirated into a new 1.5 mL centrifuge tube, and then 20 μL of 5×SDS loading buffer was added. After shaking and mixing evenly, it was boiled for 5 min to harvest the total cellular protein, and SDS-PAGE (Bio-Rad) was performed; the protein membrane was transferred (Bio-Rad) onto a PVDF membrane, blocked with 5% skim milk overnight, washed with PBST (0.05% Tween20), and then a primary antibody solution diluted in a specific ratio was added and incubated at room temperature for 1 h; the primary antibody solution was discarded, and the membrane was washed three times with PBST on a shaker at room temperature for 5 min each time. A secondary antibody solution diluted in a specific ratio was added and incubated at room temperature for 1 h; the secondary antibody solution was discarded, and the membrane was washed three times with PBST on a shaker at room temperature for 5 min each time. The PVDF membrane was placed on the operating table of the imaging system, and a hypersensitive ECL chemiluminescent solution was added dropwise onto the membrane, and the instrument parameters were adjusted for exposure. The results are as Figure 6 shown.

[0061] It can be seen from Figure 6 that the recombinant virus rH120-gD-T / B successfully expressed the gD-T / B multi-epitope protein.

[0062] Example 3 Proliferation Characteristics and Stability of Recombinant Virus rH120-gD-T / B

[0063] (1) Determination of Proliferation Curve

[0064] To determine the proliferation curve of the recombinant virus on chicken embryos, rH120-gD-T / B and the parental virus H120 strain were diluted with physiological saline and inoculated into 10-day-old SPF chicken embryos via the allantoic cavity route at an inoculum of 100 EID 50 50 , 0.1 mL / embryo. The allantoic fluid of chicken embryos was harvested at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h post-inoculation for EID 50 determination. The results are shown as Figure 7 follows.

[0065] As can be seen from Figure 7 , the growth kinetics of the recombinant virus rH120-gD-T / B in chicken embryos is similar to that of the parental virus H120 strain, and it still retains the proliferation characteristics of high-titer growth on chicken embryos.

[0066] (2) Stability detection

[0067] The recombinant virus rH120-gD-T / B was continuously passaged on chicken embryos up to 30 generations. The virus fluids of P5, P10, P15, P20, P25, and P30 generations were taken for RT-PCR identification, and the primers refer to Table 2. The results are shown as Figure 8 follows.

[0068] As can be seen from Figure 8 , bands of the expected size could be amplified from the virus rH120-gD-T / B of all passage groups, indicating that the recombinant virus can be stably passaged on chicken embryos up to at least the 30th generation.

[0069] Example 4 Viral challenge protection experiment of recombinant virus rH120-gD-T / B

[0070] Forty 1-day-old SPF chickens were randomly divided into 4 groups of 10 each, and were inoculated with the recombinant virus rH120-gD-T / B and PBS via the intranasal and intraocular routes, respectively, at an immunization dose of 10 4.0 EID 50 / chicken. Viral challenge with IBV M41 strain and ILTV WG strain was carried out at 14 days and 28 days of age, respectively. The specific grouping and experimental procedures are shown in Table 4.

[0071] Table 4 Experimental grouping and procedures

[0072]

[0073] As can be seen from Table 4, on the 14th day after immunization, a virulent M41 strain of infectious bronchitis virus was used to conduct a challenge experiment, and the chickens were continuously observed for 10 days. The morbidity rate of the challenge control group was 100%, while that of the rH120-gD-T / B immunized group was 10%. The specific manifestations of the diseased chickens were coughing, head shaking, listlessness, disheveled feathers, typical rales, and open-mouth breathing, etc. On the 28th day after immunization, a virulent WG strain of infectious laryngotracheitis virus was used to conduct a challenge experiment, and the chickens were continuously observed for 10 days. The morbidity rate of the challenge control group was 100%, while that of the rH120-gD-T / B immunized group was 0. The specific manifestations of the diseased chickens were coughing, head shaking, open-mouth breathing, hemoptysis, and death, etc.

[0074] In summary, the recombinant vaccine rH120-gD-T / B provided by the present invention can effectively resist the attacks of virulent IBV and virulent ILTV after immunizing SPF chickens, providing protection rates of 90% and 100% respectively, which proves that the bivalent vector vaccine is safe and effective.

[0075] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. ILTV gD protein antigen epitope expression cassette, characterized in that: It comprises at least one of the four ILTV antigen epitope polypeptides, and the amino acid sequences of the four ILTV antigen epitope polypeptides are respectively shown in SEQ ID NOs. 1-4.

2. The ILTV gD protein antigen epitope expression cassette according to claim 1, characterized in that: The ILTV antigen epitope polypeptide sequences are connected by a flexible linker, and the amino acid sequence of the flexible linker is shown in SEQ ID NO.

5.

3. The ILTV gD protein antigen epitope expression cassette according to claim 1, characterized in that: The sequence of the four ILTV antigen epitope polypeptides of the ILTV gD protein antigen epitope expression cassette is: FASQSTAAVTYDYILGRRALDALTIPAVGPYNRYLTRVSRGCDVVEL+GGGS+YRECGDVQLLSECAVQSAQMWALDYV+GGGS+GRFAQTALVTLEVNDRCLKIGSRLNFLPSK+GGGS+PEDVEVPEDTEHDDPNSDPDYYNDMPAVIP VEETTKSSNAVSVPIFAAFVACAVALVGLLVWSIV.

4. The ILTV gD protein antigen epitope expression cassette according to claim 1 or 2, characterized in that: The ILTV antigen epitope polypeptide comprises a T cell epitope and a B cell epitope derived from the gD protein of the WG strain of infectious laryngotracheitis virus of chicken.

5. The ILTV gD protein antigen epitope expression cassette according to claim 4, characterized in that: The T cell epitope includes an amino acid sequence such as an amino acid sequence shown in at least one of SEQ ID NOs. 6-20, and the B cell epitope includes an amino acid sequence such as an amino acid sequence shown in at least one of SEQ ID NOs. 21-25.

6. A gene, characterized in that Encoding the ILTV antigen epitope polypeptide according to claim 1.

7. A recombinant virus, characterized in that Comprising the gene according to claim 6.

8. The recombinant virus according to claim 7, characterized in that The recombinant virus uses the infectious bronchitis virus H120 strain as a skeleton, and replaces the 5ab gene of the infectious bronchitis virus H120 strain with a gene encoding an ILTV gD protein antigen epitope expression box.

9. A multi-epitope vaccine, characterized in that: Comprising the recombinant virus according to claim 7.

10. Use of the ILTV gD protein antigen epitope expression cassette according to any one of claims 1 to 5, the recombinant virus according to any one of claims 6 to 7, or the multi-epitope vaccine according to claim 8 in the preparation of or as a drug for preventing infectious bronchitis and / or infectious laryngotracheitis in chickens.

Images