Construction and application of a recombinant viral vector expressing infectious bursal disease virus VP2 protein

Abstract

The present invention constructs a recombinant turkey herpes virus expressing the VP2 gene of the infectious bursal disease virus of chickens, and the recombinant virus strain is prepared into a live vaccine, which can prevent Marek's disease and infectious bursal disease; through the homologous recombination method, the vector is used to express foreign genes, laying a foundation for using the turkey herpes virus as a vector to construct a new recombinant vaccine against Marek's disease and other pathogens.

Description

Technical Field

[0001] The present invention relates to a vaccine and method for VP2 protein of avian infectious bursal disease virus. Background Art

[0002] Without limiting the scope of the invention, the background of the invention is described in conjunction with the morbidity and mortality of the infectious bursal disease (IBD) virus in chickens. Infectious bursal disease in chickens is an acute, highly contagious, infectious disease caused by infectious bursal disease virus (IBDV). The disease can cause immunosuppression in the body by damaging the bursa of Fabricius, thereby causing high mortality in chicks. There are two known serotypes of IBDV, namely serotype I and type II, of which serotype II is non-pathogenic or has very weak pathogenicity, and serotype I can cause disease in chickens. In the decades since the discovery of the disease, serotype I IBDV has mutated, and classic strains, variant strains and super-strong strains have appeared one after another. Since the 1990s, very virulent IBDV (vvIBDV) with a high mortality rate has spread globally, causing huge losses to the poultry industry. Due to the development and use of vaccines and the development of breeding management technology, the prevalence of vvIBDV has been gradually controlled. However, new changes have occurred in IBD in recent years. Atypical IBD has been found in some chicken farms in my country. Infected chickens have no obvious external symptoms, but their central immune organ, the bursa of Fabricius, is severely atrophied, leading to severe immunosuppression and decreased production performance, posing a serious threat to the healthy development of the poultry industry.

[0003] Vaccination is the most important measure to prevent and control IBD. Currently, commercialized live vaccines for infectious bursal disease are divided into three categories: attenuated, moderately virulent, and highly virulent. Attenuated vaccines are safe for chickens, but the immune protection effect on chickens with maternal antibodies is not ideal. Although the immune protection of moderately virulent and highly virulent vaccines is better, they have a certain pathogenicity that can damage the bursa of Fabricius and there is also a risk of virulence reversion. In addition, due to the interference of maternal antibodies and the characteristics of IBDV, traditional vaccines can form an inevitable immune blank period in practical applications. VP2 protein is the main protective antigen of IBDV and its neutralizing antibodies can trigger the body's protection against bursal virulent attacks. Therefore, constructing a vaccine expressing the IBDV VP2 gene has good application value.

[0004] Turkey Herpesvirus (HVT) is an alpha herpesvirus belonging to Marek's disease virus serotype III. It is a virus that is common in turkeys and non-pathogenic to poultry. Since the 1970s, HVT Fc-126 strain has been used as a vaccine for the prevention and control of Marek's disease. Due to its non-pathogenicity to poultry, HVT has become one of the key research directions as a live virus vector to construct a new generation of safe and effective vaccines. HVT has more than 20 replication non-essential regions for the insertion of exogenous genes and can insert longer exogenous genes. Summary of the invention

[0005] On the one hand, the present invention provides a recombinant vector of the VP2 gene of avian infectious bursal disease virus or a recombinant vector of an immunogenic derivative of the VP2 gene, wherein the VP2 gene is derived from a protective antigen epitope gene of infectious bursal disease virus.

[0006] Furthermore, the VP2 gene sequence is encoded by SEQ ID No: 1, and the immunogenic derivative refers to a gene sequence that differs from the VP2 sequence by 1 to 100 nucleotides, preferably a gene sequence that differs by 1 to 50 nucleotides, or a nucleotide sequence that contains the VP2 gene.

[0007] In a specific embodiment, the recombinant vector of the VP2 gene of avian infectious bursal disease virus or the recombinant vector of its immunogenic derivative gene is encoded by a viral vector. Further, the viral vector is turkey herpes virus.

[0008] In another specific embodiment, the recombinant vector of the VP2 gene of avian infectious bursal disease virus or its immunogenic derivative gene is a recombinant virus (rHVT-U3VP2), which is a recombinant virus (rHVT-U3VP2) composed of the non-pathogenic backbone of turkey herpes virus and the VP2 gene of avian infectious bursal disease virus or its immunogenic derivative gene.

[0009] In another aspect, the present invention provides a vaccine composition comprising a recombinant vector of the VP2 gene of avian infectious bursal disease virus or a recombinant vector of an immunogenic derivative of the VP2 gene.

[0010] In one embodiment, the vaccine composition comprises a recombinant virus (rHVT-U3VP2) in which the non-pathogenic backbone of turkey herpesvirus is combined with the VP2 gene of avian infectious bursal disease virus or its immunogenic derivative gene. Further, the vaccine composition comprises a recombinant virus (rHVT-U3VP2) of turkey herpesvirus encoding the VP2 protein or its immunogenic derivative protein.

[0011] Furthermore, the vaccine is a live vaccine or a modified live vaccine.

[0012] In another aspect, the vaccine composition of the present invention further comprises an adjuvant.

[0013] In one embodiment, the adjuvant is selected from the group consisting of: oil-in-water adjuvants, polymer and water adjuvants, water-in-oil adjuvants, aluminum hydroxide adjuvants, vitamin E adjuvants, and combinations thereof.

[0014] In a specific embodiment, the adjuvant comprises an oil emulsion comprising polyoxyethylene-polyoxypropylene block copolymer, squalane, polyoxyethylene sorbitan monooleate, and buffered saline (SP-oil).

[0015] In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0016] In another embodiment, the vaccine composition further comprises at least one additional antigen.In certain embodiments, the at least one additional antigen protects against microorganisms that can cause avian disease.

[0017] In another embodiment, the antigens include one or more antigens derived from bacteria, viruses, or parasitic microorganisms known to be pathogenic in poultry.

[0018] The present invention also provides a method for protecting poultry from Marek's disease and infectious bursal disease virus infection. The method comprises administering to poultry an immunologically effective amount of a vaccine composition disclosed herein, or a recombinant viral vector. In various embodiments, the method comprises administering to poultry a vaccine composition or a recombinant viral vector by one or more than one route selected from parenteral, oral, intradermal, and transdermal. In another embodiment, the vaccine composition or recombinant viral vector is administered in a single dose.

[0019] In another embodiment, the vaccine composition of the invention is administered in combination with at least one additional antigen that protects against microorganisms that can cause disease in avian species. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Schematic diagram of the transfer vector p19-UL3 / 4-RFP.

[0021] Figure 2 : Schematic diagram of the transfer vector p19-UL3 / 4-VP2-G.

[0022] Figure 3 : PCR identification of recombinant virus rHVT-U3VP2.

[0023] Figure 4 : Indirect immunofluorescence identification of recombinant virus rHVT-U3VP2.

[0024] Figure 5: Genetic stability test of recombinant virus rHVT-U3VP2.

[0025] Figure 6 : Monoclonal antibody stability test.

[0026] Figure 7 :Identification of monoclonal antibody 44-10, 115-5 epitopes and sequence analysis of the epitope recognized by cloned antibody 44-10

[0027] Figure 8 :Infectious bursal disease experiment P24 negative control and BVDV positive control diagram DETAILED DESCRIPTION

[0028] Although the making and use of various embodiments of the present invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be used in various specific situations. The specific embodiments discussed here only illustrate the specific ways to make and use the present invention, and do not limit the scope of the present invention.

[0029] Provided herein are VP2 immunogenic derivative genes obtained by DNA modification of the VP2 gene or the VP2 gene.

[0030] In one embodiment, when the VP2 gene or a VP2 immunogenic derivative gene is placed in a herpesvirus of turkeys (HVT) backbone, the chimeric virus induces high neutralizing protective antibody titers against the VP2 gene.

[0031] Furthermore, the present invention first constructs an HVT virus with a red fluorescent protein gene (RFP) marker inserted between the UL3 and UL4 genes of HVT, then constructs a transfer vector carrying a green fluorescent protein gene (GFP) screening marker, an IBDV VP2 gene and a loxp gene, and applies the green fluorescent protein screening technology to obtain a recombinant turkey herpes virus strain (rHVT-U3VP2) expressing the IBDV VP2 gene. The double-marker gene screening technology of the present invention improves the screening efficiency of the recombinant virus; and the recombinant virus can completely protect chickens from the attack of infectious bursal disease virus after immunizing them.

[0032] To facilitate understanding of the present invention, a number of terms are defined below. The terms defined herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention relates.

[0033] The term "antigen" refers to a compound, composition or immunogenic substance that can stimulate the production of antibodies or T cell responses or both in an animal, including compositions that are injected or absorbed into an animal. An immune response can be generated to an entire molecule or a portion of a molecule (e.g., an epitope or hapten).

[0034] The term "epitope" refers to a discrete portion of an antigen that serves as a binding site recognized by an antibody or T cell receptor. For example, an epitope can refer to an antigenic determinant cluster recognized by the complementary determining region (CDR) of an antibody. The site of each binding antibody that binds to the epitope is called a "paratope."

[0035] "Immunogenic composition or immunological composition" refers to a composition of matter comprising at least one antigen that elicits a cellular and / or antibody-mediated immune response in a host to the composition or vaccine of interest.

[0036] The term "immune response" refers to a response elicited in an animal. An immune response may refer to cellular immunity (CMI) or humoral immunity or may involve both. The present invention also contemplates responses that are limited to a portion of the immune system. Typically, an "immune response" includes, but is not limited to, the production or activation of antibodies, B cells, helper T cells, suppressive T cells, and / or cytotoxic T cells directed specifically to an antigen contained in a composition or vaccine of interest. Preferably, the host will exhibit a therapeutic or protective immune response, thereby enhancing resistance to new infections and / or reducing the clinical severity of the disease. This protection is demonstrated by a reduction or lack of symptoms typically displayed by an infected host, a faster recovery time in an infected host, and / or lower viral titers.

[0037] The term "antibody" includes intact immunoglobulin molecules as well as parts, fragments, and derivatives thereof, such as Fab, Fab', F(ab')2, Fv, Fsc, CDR regions, or any part of an antibody capable of binding to an antigen or epitope, including chimeric antibodies with bispecificity or chimeric antibodies in which an antigen-binding domain derived from an antibody is combined with another polypeptide.

[0038] The term "monoclonal antibody" includes chimeric antibodies and fragments, portions, regions or derivatives thereof derived from monoclonal antibodies and corresponding monoclonal antibodies of various species provided by any known technique, including but not limited to molecular techniques, including phage display, affinity maturation and site-directed mutagenesis, and chemical techniques including covalent modification and conjugation.

[0039] As used herein, "adjuvant" refers to a composition consisting of one or more than one substance that enhances the immune response to an antigen. The mechanism by which adjuvants work is not fully understood. Some adjuvants are thought to enhance the immune response by slowly releasing the antigen, while other adjuvants themselves have strong immunogenicity and are thought to act synergistically.

[0040] As used herein, the term "multivalent" refers to a vaccine containing more than one antigen, whether from the same species (ie, different isolates of IBDV), from different species or referring to a vaccine containing a combination of antigens from different genera.

[0041] As used herein, the term "avian" refers to animals of animal origin, which includes chickens.

[0042] As used herein, the term "virulence" refers to the ability of an isolate to retain its infectivity in an animal host.

[0043] The pathogen can be a bacterium, a virus, a protozoa, or a fungus. Inactivation can be accomplished by a variety of methods, including freeze-thaw, chemical treatment (e.g., with thimerosal or formalin), sonication, radiation, heating, or any other conventional method sufficient to prevent replication or growth of the organism while maintaining its immunogenicity.

[0044] The term "pharmaceutically acceptable carrier" refers to a fluid carrier for containing a vaccine antigen that can be injected into a host without side effects. Suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, sterile water, saline, glucose, dextrose, or a buffered solution. The carrier may include adjuvants, including, but not limited to, diluents, stabilizers (sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffers, viscosity enhancing additives, colorants, and the like.

[0045] The term "vaccine composition" includes at least one antigen or immunogen in a pharmaceutically acceptable carrier, which can be used to induce an immune response in a host. The vaccine composition can be administered in doses and by techniques well known to medical or veterinary technicians, taking into account factors such as the age, sex, weight, species and condition of the recipient animal, and the route of administration. The route of administration can be transdermal (through the skin or through the oral, nasal, anal, vaginal mucosa via intradermal, transdermal, subcutaneous, intramuscular routes) or by parenteral routes (intravenous or intraperitoneal). The vaccine composition can be administered alone, or can be co-administered or sequentially administered with other treatments or therapies. The administration form can include suspensions, syrups or elixirs, and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injection administration), such as sterile suspensions or emulsions. The vaccine composition can be administered in a spray form or mixed in food and / or water or mixed with suitable carriers, diluents, or excipients such as sterile water, saline, glucose, etc. for delivery. The composition may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, etc., depending on the route of administration and the desired formulation.

[0046] Information on microbial resources involved in the present invention

[0047] The microorganism involved in the present invention is: Turkey Herpesvirus (HVT) CVCC AV19 strain, which is stored in the China Veterinary Drug Administration, China Veterinary Microbiological Culture Collection Center, No. 33, Qingfeng Road, Biomedicine Base, Daxing District, Beijing; by molecular biotechnology, the VP2 gene of infectious bursal disease virus of chicken is inserted between the UL3 gene and the UL4 gene of HVT, and the recombinant virus obtained is named rHVT-U3VP2. The strain of virus was submitted to the General Microbiology Center of China Microbiological Culture Collection Committee for preservation on June 16, 2020. The address of the preservation unit is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is: CGMCC No. 19996; the classification name of the preserved biological material is: rHVT-U3VP2, and it is proved to be in a viable state by the preservation unit.

[0048] Example

[0049] The following examples are provided to better illustrate the technical solution of the present invention, but do not limit the technical solution of the present invention. Example 1: Construction of recombinant turkey herpes virus (rHVT-U3VP2 strain)

[0050] The construction method of the recombinant turkey herpes virus (rHVT-U3VP2 strain) of the present invention is as follows:

[0051] (1) Construction of transfer vector containing RFP expression cassette

[0052] pUC19 vector, used for cloning, sequencing and construction of transfer vector, was purchased from Takara Biotechnology (Dalian) Co., Ltd. The RFP reporter gene vector p19-UL3 / 4-RFP containing pHCMV promoter and BGH ployA (see Figure 1 ), constructed and preserved by the laboratory of the inventor.

[0053] (2) Primer design

[0054] The primer design sequences of the present invention are shown in Table 1

[0055] Table 1: Information of relevant primers involved in the construction of the present invention

[0056]

[0057] (3) Construction of RFP-tagged turkey herpesvirus rHVT-U3R

[0058] 1) Construction of transfer vector p19-UL3 / 4-RFP

[0059] According to the gene sequence of HVT Fc-126 strain in GenBank (Genbank accession number AF291866), CEDesign V1.04 was used to design homologous recombination primers UL3 F / R and UL4 F / R. The genomic DNA of turkey herpesvirus Fc-126 strain (CVCCAV19) was used as a PCR amplification template to amplify the UL3 gene and UL4 gene, respectively. The amplified target fragments were connected to the pUC19 vector to construct the recombinant plasmid pUC19-UL3 / 4. CE Design V1.04 was used to design homologous recombination primers pHCMV~RFP F / R and BGH ployA F / R. The plasmids pT-TK-RFP and pT-BGH ployA in the laboratory were used as templates to amplify pHCMV~RFP and BGH ployA fragments, respectively. The amplified target fragments were connected to pUC19-UL3 / 4 to obtain the transfer vector p19-UL3 / 4-RFP.

[0060] 2) Homologous recombination

[0061] According to Lipofectamine TM According to the instructions of the 3000 transfection kit (Invitrogen), the transfer vector p19-UL3 / 4-RFP and HVT Fc-126 strain were transfected into chicken embryo fibroblasts (CEF). The specific steps are as follows:

[0062] When the CEF in the six-well plate grows to a 70% to 90% cell monolayer, inoculate an appropriate amount of HVT Fc-126 strain and place it in a 37°C incubator with 5% CO2 for 12 hours; take two clean centrifuge tubes, add 125μL Opti-MEM culture medium to each tube; add 7.5μL of lipofectamine 3000 reagent to one tube and shake to mix; add 10μL of P3000 reagent and 2.5μg of p19-UL3 / 4-RFP transfer vector to the other tube, shake to mix; mix the prepared reagents in the two tubes, mix well, and let stand at room temperature for 15 minutes; take out the CEF inoculated with HVT in the 6-well plate from the incubator, discard the culture medium, and wash twice with Opti-MEM culture medium; evenly drop the reagent after standing in one well of the 6-well plate, add 500μL Opti-MEM medium was placed in a 37°C incubator with 5% CO2 for 8-10 hours; the liquid in the transfected wells on the cell plate was discarded and replaced with 5% fetal bovine serum F-10 medium for continued culture. The transfected cells were observed every 12 hours, and the cells were harvested when they were completely diseased. The recombinant virus obtained initially was inoculated into 6-well CEF cells that had grown into a good cell monolayer and continued to be cultured. The cells were observed under a fluorescence microscope every 24 hours, and the plaques that emitted red fluorescence were picked and inoculated into 6-well CEF cells that had grown into a good cell monolayer. Plaque purification was performed 3 to 4 times, and the recombinant virus obtained was named rHVT-U3R.

[0063] (4) Construction of a recombinant turkey herpes virus expressing the infectious bursal disease virus VP2 gene (rHVT-U3VP2 strain)

[0064] 1) Construction of transfer vector p19-UL3 / 4-VP2-G

[0065] Extract RNA from IBDV HB96 strain, and amplify VP2 gene by RT-PCR with primers VP2F and VP2R. Double digest p19-UL3 / 4-RFP with BamH I and NheI, then connect the target fragment with the digested p19-UL3 / 4-RFP vector to obtain recombinant plasmid pUC19-UL3 / 4-VP2. Digest plasmid pGFP-loxp and recombinant plasmid pUC19-UL3 / 4-VP2 with BamH I, then connect to obtain recombinant transfer vector p19-UL3 / 4-VP2-G (see Figure 2 ).

[0066] 2) Homologous recombination

[0067] According to Lipofectamine TM3000 transfection kit instructions, transfer vector p19-UL3 / 4-VP2-G and rHVT-U3R virus to transfect chicken embryo fibroblasts (CEF). The specific steps are as follows:

[0068] When the CEF in the six-well plate grows to a 70% to 90% cell monolayer, inoculate an appropriate amount of rHVT-U3R and place it in a 37°C incubator with 5% CO2 for 12 hours; take two clean centrifuge tubes, add 125μL Opti-MEM culture medium to each tube; add 7.5μL of lipofectamine 3000 reagent to one tube and shake to mix; add 10μL of P3000 reagent and 2.5μg of p19-UL3 / 4-VP2-G transfer vector to the other tube, shake to mix; mix the prepared reagents in the two tubes, mix well, and let stand at room temperature for 15 minutes; take out the CEF inoculated with rHVT-U3R in the 6-well plate from the incubator, discard the culture medium, and wash twice with Opti-MEM culture medium; evenly drop the reagent after standing in one well of the 6-well plate, add 500μL Opti-MEM medium was placed in a 37°C incubator with 5% CO2 for 8-10 hours; the liquid in the transfected wells on the cell plate was discarded and replaced with 5% fetal bovine serum F-10 medium for continued culture. The transfected cells were observed every 12 hours, and the cells were harvested when they were completely diseased. The recombinant virus obtained initially was inoculated into 6-well CEF cells that had grown into a good cell monolayer and continued to be cultured. The cells were observed under a fluorescence microscope every 24 hours, and the plaques that emitted green fluorescence were picked and inoculated into 6-well CEF cells that had grown into a good cell monolayer. Plaque purification was performed 3 to 4 times, and the recombinant virus obtained was named rHVT-U3VP2-G.

[0069] 3) Knockout of GFP gene in rHVT-U3VP2-G

[0070] The obtained recombinant virus rHVT-U3VP2-G was inoculated into 6-well CEF cells that had grown into a good cell monolayer, and placed in a 37°C incubator with 5% CO2 for 12 hours; two clean centrifuge tubes were taken, and 125 μL Opti-MEM culture medium was added to each tube; 7.5 μL of lipofectamine 3000 reagent was added to one tube, and oscillated to mix; 10 μL of P3000 reagent and 2.5 μg of pcDNA3.1-Cre plasmid were added to the other tube, and oscillated to mix; the prepared reagents in the two tubes were mixed, mixed thoroughly, and allowed to stand at room temperature for 15 minutes; the CEF inoculated with rHVT-U3VP2-G in the 6-well plate was taken out of the incubator, the culture medium was discarded, and it was washed twice with Opti-MEM culture medium; the reagent after standing was evenly dropped into one well of the 6-well plate, and 500 μL Opti-MEM medium was placed in a 37°C incubator with 5% CO2 for 8-10 hours; the liquid in the transfected wells on the cell plate was discarded and replaced with 5% fetal bovine serum F-10 medium for continued culture. The transfected cells were observed every 12 hours, and the cells were harvested when they were completely diseased. The recombinant virus obtained initially was inoculated into 6-well CEF cells that had grown into a good cell monolayer and continued to be cultured. The cells were observed under a fluorescence microscope every 24 hours, and the non-fluorescent plaques were picked and inoculated into 6-well CEF cells that had grown into a good cell monolayer. Plaque purification was performed in this way until no fluorescent plaques were observed under a fluorescence microscope. The obtained recombinant virus was named rHVT-U3VP2.

[0071] Example 2: Identification of recombinant turkey herpes virus (rHVT-U3VP2 strain)

[0072] 1. PCR identification of recombinant virus rHVT-U3VP2

[0073] PCR amplification was performed using primers UL3F and UL4R, and sequencing of the PCR product confirmed that the VP2 gene was successfully inserted between the UL3 and UL4 genes (see Figure 3 ).

[0074] 2. Indirect immunofluorescence identification of recombinant virus rHVT-U3VP2

[0075] The recombinant virus rHVT-U3VP2 and its parent virus were inoculated into CEF, and when the cytopathic effect reached 80%, the expression of VP2 gene was detected by indirect immunofluorescence method (see Figure 4 ).

[0076] Indirect immunofluorescence steps: discard the cell culture medium, wash the cell surface lightly with PBS (pH7.2) once, then add 500 μL of 80% pre-cooled acetone solution to each well, place at room temperature for 20 minutes, discard the liquid, and dry naturally; wash the cell surface once with PBS (pH7.2), then add 300 μL of mouse anti-IBDV VP2 monoclonal antibody at working concentration, and incubate in a 37°C incubator for 30 minutes; discard the liquid, wash 3 times with PBS (pH7.2) containing 0.05% Tween-20; then add 300 μL of Cy3-labeled goat anti-mouse IgG conjugate to each well, incubate in a 37°C incubator for 30 minutes; wash 3 times with PBS (pH7.2) containing 0.05% Tween-20; observe under a fluorescent inverted microscope. After staining with mouse anti-IBDV VP2 monoclonal antibody, the recombinant virus rHVT-U3VP2 showed specific red fluorescence, while the parent virus had no fluorescence. The results showed that the recombinant virus expressed VP2 protein in CEF.

[0077] 3. Determination of the virus content of recombinant virus rHVT-U3VP2

[0078] The recombinant virus rHVT-U3VP2 was inoculated into CEF, and the plaque count was performed using CEF. The result was 10 6.0 PFU / 0.1ml.

[0079] 4. Genetic stability test of recombinant virus rHVT-U3VP2

[0080] The recombinant virus rHVT-U3VP2 was propagated on CEF cells for 20 generations. The 1st, 5th and 10th generations of the virus were tested by PCR and indirect immunofluorescence. The results showed that the obtained recombinant virus rHVT-U3VP2 was stably inherited and correctly expressed VP2 protein in CEF (see Figure 5 ).

[0081] 5. Animal testing

[0082] 1) Safety test

[0083] 25 one-day-old SPF chickens were injected subcutaneously with recombinant virus rHVT-U3VP2 in the neck, 0.2ml / chicken (20000PFU / ml) and observed for 21 days. All chickens were killed and dissected. No gross lesions of infectious bursal disease were found in all chickens. Another 30 one-week-old SPF chickens were randomly divided into 3 groups, 10 chickens in each group. The first group was injected subcutaneously with recombinant virus rHVT-U3VP2 in the neck as the immunization group, 0.2ml / chicken (20000PFU / ml); the second group was injected subcutaneously with HVT Fc-126 strain (CVCC AV19) in the neck as the parent strain control group, 0.2ml / chicken (20000PFU / ml); the third group was not treated and served as the blank control group. Each group was isolated and raised separately. Observation continued for 120 days. The chickens in the immunized group, parental strain control group, and blank control group did not show any clinical symptoms of Marek's disease; at the end of the observation, all chickens were autopsied and macroscopic lesions of Marek's disease were observed. The results showed that the recombinant virus rHVT-U3VP2 was as safe for SPF chickens as the parental strain HVT Fc-126.

[0084] 2) Immunogenicity test

[0085] ① Marek's disease section

[0086] Sixty one-day-old SPF chickens were used, of which 20 were injected intramuscularly with the recombinant virus rHVT-U3VP2 as the immunization group, 0.2 ml / chicken (1000 PFU / ml); the other 20 were not immunized as the challenge control group; the remaining 20 were not immunized and challenged as the negative control. 21 days after vaccination, all immunized chickens and challenge control chickens were intraperitoneally inoculated with 10 -1 0.2ml, and observed for 60 days. The results showed that 19 chickens in the challenge control group became ill, with a 95% MD positive rate. The autopsy showed obvious Marek's macroscopic lesions. There were no Marek's macroscopic lesions in the immunized group of 16 chickens, with 80% protection. There were no MD lesions in the healthy group.

[0087] ② Infectious bursal disease (IBDV VP2 protein)

[0088] Thirty one-week-old SPF chickens were randomly divided into three groups, with 10 in each group. The first group was injected subcutaneously with the recombinant virus rHVT-U3VP2 in the neck as the immunization group, 0.2 ml / bird (5000 PFU / ml); the second group was not immunized and used as the challenge control; the third group was not immunized and used as the blank control. Each group was isolated and raised separately. 28 days after immunization, blood was collected from the wing vein and IBDV BC6 / 85 strain (CVCCAV7) was inoculated intranasally, 100 BID per chicken. After 3 days of observation, all experimental chickens in the immunization group and the control group were killed, dissected and the bursa of Fabricius was removed to observe the bursal lesions. The bursa of the blank control group was normal, the bursa of the challenge control group was 100% lesional, and the immunization group was 100% protected, indicating that rHVT-U3VP2 has good immunogenicity.

[0089] Example 3: Preparation of vaccine

[0090] (1) Vaccine preparation

[0091] 1) Cell preparation: Select well-developed 9- to 11-day-old SPF chicken embryos, prepare CEFs by conventional trypsin digestion method, and culture for about 24 hours to grow into a monolayer for later use.

[0092] 2) Preparation of virus solution: Inoculate the production virus rHVT-U3VP2 strain into CEF cell monolayer at a volume ratio of 0.01% to 0.5%, and culture in a 5% CO2 37°C incubator for 36 to 72 hours. When the pathological rate reaches more than 85%, harvest the cells and add cell freezing solution to prepare a cell suspension.

[0093] 3) Preparation and packaging: Add 10% DMSO (V / V) and 20% bovine serum (V / V) into the collected cell suspension, mix well, and package and seal 2.0 ml per ampoule.

[0094] 4) Storage: Immediately freeze the packaged and labeled vaccine ampoules in a programmed freezer. Take them out and put them in a liquid nitrogen tank.

[0095] Example 4: Vaccine Testing

[0096] (1) Vaccine testing:

[0097] The relevant inspection methods involved in the present invention are carried out according to the methods of the Veterinary Pharmacopoeia of the People's Republic of China (China Veterinary Pharmacopoeia Committee. Veterinary Pharmacopoeia of the People's Republic of China 2015 Edition (Three Volumes). China Agriculture Press, 2016, the present invention is referred to as "China Veterinary Pharmacopoeia").

[0098] 1) Appearance: Light pink suspension.

[0099] 2) Sterility test is carried out according to the current "Chinese Veterinary Pharmacopoeia" and sterile growth is prohibited.

[0100] 3) Mycoplasma test was carried out according to the current "Chinese Veterinary Pharmacopoeia", and no mycoplasma growth was found.

[0101] 4) Exogenous virus test

[0102] a. Chicken infectious anemia virus (CAV) test: 20 one-day-old SPF chickens were inoculated with 10 doses of vaccine by eye and nose drops, and 100 doses of vaccine were injected intramuscularly. After 21 days, the inoculation was repeated once according to the above method and dosage. Blood was collected 42 days after the first inoculation, and serum was separated and tested for CAV antibodies using a commercial CAV antibody detection kit, which was negative.

[0103] b. Other exogenous virus tests are carried out in accordance with the current "Chinese Veterinary Pharmacopoeia" and there is no exogenous virus contamination.

[0104] 5) Identification test

[0105] After the vaccine was diluted with vaccine diluent, an indirect immunofluorescence reaction was performed with mouse anti-IBDV VP2 monoclonal antibody. The CEF culture dish inoculated with the vaccine should show red fluorescence, while the HVT virus control culture dish and the cell control culture dish did not show this fluorescence reaction. According to the standard test, the CEF culture dish inoculated with the vaccine showed red fluorescence, while the HVT virus control culture dish and the cell control culture dish did not show this fluorescence reaction.

[0106] 6) Safety test: 20 one-day-old SPF chickens were used, 10 of which were injected intramuscularly or subcutaneously with 0.2 ml of vaccine (containing 10 doses), and the other 10 were used as controls. After 21 days of observation, at least 8 control chickens should survive, and the number of non-specific deaths in the immunized group should not exceed the standard test of the control group. The immunized group: 10 / 10 survived, with no clinical symptoms of MD and visual changes; the control group: 10 / 10 survived.

[0107] 7) Effectiveness test

[0108] Immune attack method:

[0109] ① 60 one-day-old SPF chickens were used, of which 20 were injected subcutaneously in the neck with 1 dose of vaccine (0.2 ml / chicken) as the immunization group, and the other 20 were not immunized as the challenge control group; the remaining 20 were not immunized and challenged as the negative control. Ten days after vaccination, all immunized chickens and challenge control chickens were intraperitoneally inoculated with the super-virulent Jing-1 strain of Marek's disease virus for 10 -1 0.2ml, observe for 60 consecutive days. Chickens that died during the observation period need to be autopsied to see if there are gross lesions of Marek's disease in chickens; at the end of the observation, the test chickens will be killed and autopsied to see if there are gross lesions of Marek's disease in chickens. The MD positive rate of the challenge control chickens is ≥60%, the MD reduction rate of the immunized group is ≥60%, and the 20 negative control chickens raised in isolation in the same batch should have no MD lesions.

[0110] The results showed that the MD positive rate of the challenged control chickens was 80%, and the MD reduction rate of the immunized group was 81%. 20 / 20 of the negative control chickens survived and had no MD lesions.

[0111] ②Infectious bursal disease (IBDV VP2 protein)

[0112] Take 10 1-week-old SPF chickens, mark the doses on the bottle label, inject 0.2 ml of vaccine (containing 10 doses) into the neck of each chicken, and take 5 chickens each as non-immunized control and blank control, and isolate and raise them under the same conditions. After 21-28 days, take all immune chickens together with control chickens, and each chicken is attacked with IBDV BC6 / 85 strain (CVCC AV7) virulent at least 10 BID in the eyes. After 72 hours, all chickens are dissected to check the changes in the bursa of Fabricius. There should be no changes in the bursa of Fabricius of the 5 chickens in the blank control group, and at least 4 of the 5 chickens in the challenge control group should have lesions in the bursa of Fabricius, and at least 8 chickens in the immune group should have no lesions in the bursa of Fabricius.

[0113] The results showed that 5 / 5 of the blank control group had no bursal lesions, 5 / 5 of the challenge control group had bursal lesions, and 10 / 10 of the immunized group had no bursal lesions.

[0114] Example 5: Application of recombinant virus rHVT-U3VP2

[0115] According to the HA gene sequence of H9 subtype avian influenza virus published by GenBank, an artificially synthesized HA gene (SEQ ID No: 2) was inserted into the transfer vector p19-UL3 / 4-VP2-G by PstI digestion to obtain the recombinant transfer vector p19-UL3 / 4-HA-G.

[0116] According to Lipofectamine TM According to the instructions of the 3000 transfection kit, the transfer vector p19-UL3 / 4-HA-G and rHVT-U3R virus were transfected into chicken embryo fibroblasts (CEF). After plaque purification, the recombinant virus rHVT-U3HA-G strain was obtained. The recombinant virus rHVT-U3HA was obtained using the Cre-loxp system.

[0117] The invention constructs a recombinant turkey herpes virus (rHVT-U3VP2) expressing the infectious bursal disease virus VP2 gene of chickens, and the recombinant virus rHVT-U3VP2 strain is prepared into a live vaccine, which can prevent Marek's disease and infectious bursal disease; during the construction process, a recombinant turkey herpes virus vector (rHVT-U3R) carrying a red fluorescent marker can be used to express exogenous genes through a homologous recombination method, thereby laying a foundation for constructing a new recombinant vaccine against Marek's disease and other pathogens using the turkey herpes virus as a vector.

Claims

1. A recombinant turkey herpes virus expressing the infectious bursal disease virus VP2 gene, characterized in that: The deposit number of the recombinant turkey herpes virus is CGMCC No.19996.

2. The recombinant turkey herpes virus according to claim 1, characterized in that The recombinant turkey herpes virus is a recombinant virus obtained by inserting the VP2 gene between the UL3 gene and the UL4 gene of the turkey herpes virus, and the VP2 gene sequence is SEQ ID No.

1.

3. A vaccine composition, characterized in that: The vaccine composition comprises the recombinant turkey herpes virus as described in claim 1.

4. The vaccine composition according to claim 3, characterized in that The vaccine composition also includes an adjuvant.

5. Use of the vaccine composition according to any one of claims 3 to 4 in the preparation of a preparation for preventing or treating Marek's disease and infectious bursal disease in chickens.

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