Recombinant turkey herpesvirus expressing fowl adenovirus serotype 4 fiber protein, uses thereof, and vaccine

Abstract

The present invention belongs to the field of biotechnology and discloses two recombinant turkey herpesviruses expressing the fiber protein of fowl adenovirus serotype 4. A target gene is inserted between the HVT053 site and the HVT054 site or between the HVT087 site and the HVT088 site of the turkey herpesvirus, and the target gene is the Fiber-2 gene with a promoter and a terminator to obtain a recombinant virus; the nucleotide sequence of the Fiber-2 gene is as shown in SEQ ID NO.1. The replication ability of the recombinant virus is consistent with that of the parental virus, and it can provide good immune protection against FADV-4 virus, meeting the requirements of a vaccine candidate strain. At the same time, the present invention also provides the target gene, uses and vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a recombinant turkey herpesvirus expressing fowl adenovirus serotype 4 spike protein, uses thereof, and a vaccine. Background Art

[0002] Fowl adenovirus (FADV) is an unenveloped virus with double-stranded DNA, belonging to the family Adenoviridae and the genus Aviadenovirus. According to restriction endonuclease digestion patterns and serum cross-neutralization tests, FADV is divided into 5 species (FADV-A to FADV-E) and 12 serotypes (FAdV-1 to 8a and 8b to 11). Hydropericardium syndrome (HHS) is an acute and highly contagious disease caused by fowl adenovirus serotype 4 (FADV-4), which is prevalent in chicken flocks and causes huge economic losses. The main structural proteins of FADV serotype 4 are: hexon, penton, and fiber protrusions located on the penton. There are 2 spike proteins on each penton, namely Fiber-1 and Fiber-2. The spike protein carries subgroup-specific and type-specific antigenic epitopes, which play an extremely important role in initiating virus infection and inducing neutralizing antibodies, and can be used as an effective protective immunogen for developing FADV vaccines. Many studies have shown that recombinant Fiber-2 protein can better resist the lethal attack of FADV serotype 4 than other capsid proteins (including Fiber-1, Penton, and Hexon), indicating that the Fiber-2 gene has good immunogenicity.

[0003] HHS is mainly caused by highly pathogenic FADV serotype 4 and is a high-mortality disease mainly infecting broiler chickens, characterized by the accumulation of clear straw-colored fluid in the pericardial sac, as well as enlarged and discolored livers with hemorrhage and / or nephritis lesions. After 2015, the outbreak of FADV serotype 4 in China has caused huge economic losses to the domestic poultry breeding industry, and now this disease has caused epidemics in many places in China. The latest systematic epidemiological surveys all show that FADV serotype 4 is preponderantly prevalent in poultry. FADV serotype 4 mainly affects young chicken flocks, especially broiler chickens in the growing period of 3 - 8 weeks of age, and spreads through vertical and horizontal routes. Moreover, FADV serotype 4 can be effectively transmitted among chickens through aerosols, seriously threatening the healthy development of China's poultry industry. Some studies have shown that FADV serotype 4 has an infective tropism for lymphoid tissues, and it is found that the infection of FADV serotype 4 can affect the lymphocyte subsets of SPF chickens, resulting in a decrease in B cells and T cells in the lymphoid organs of SPF chickens. At the same time, mixed infections of FADV serotype 4 with immunosuppressive diseases such as IBV, AIV-H9, IBDV, and chicken infectious anemia have been found clinically, causing severe immunosuppression in poultry.

[0004] At present, traditional inactivated vaccines are mainly used to prevent and control hepatitis hydropericardium syndrome at home and abroad. The inactivated vaccines are mostly prepared from viruses proliferated by LMH cells. Such inactivated vaccines may have the risk of incomplete inactivation, resulting in a great risk of virus dissemination in the immunized chicken flock. Moreover, since inactivated vaccines cannot replicate in the body, the immune response induced in the body is short-lived, and multiple immunizations are required to achieve the immune effect. Different from inactivated vaccines, live vector vaccines can carry foreign genes for transcription and expression in the body, thereby stimulating the body to produce corresponding cellular immunity. HVT is non-pathogenic to chickens but can induce viremia, so it can induce a protective immune response against MDV. Moreover, this virus can persistently infect chickens, stimulating the body to continuously produce antibodies, and a single vaccination can achieve lifelong immunity. Therefore, under the current large-scale farming conditions, for these two diseases that pose great harm to the poultry industry, it is necessary to develop effective vaccines to combat MD and HHS simultaneously.

[0005] The following prior art is available regarding the recombinant virus of Fiber-2:

[0006] Prior art 1: CN110484515A discloses a vaccine vector for preventing FAdV-4 and NDV, its preparation method and application; it is recorded in paragraph 124 of its specification that existing studies have shown that the Fiber 2 protein of FAdV-4 is a decisive protein determining the virulence of different strains of FAdV-4. The study in this case also found that the virus proliferation ability of the NDV virus expressing the Fiber 2 protein was significantly lower than that of the parental strain, the virulence was higher than that of the parental strain, and its immune protection effect was also lower than that of the recombinant Newcastle disease virus expressing the Fiber-1 protein of avian adenovirus type 4 constructed in this case.

[0007] It is recorded in paragraph 150 of this case that in the first immunization challenge test, the mortality rate of the Fiber 1 group was 40%, the mortality rate of the Fiber 2 group was 50%, and the mortality rates of the LaSota group and the PBS group were 90% and 90% respectively; in the second immunization challenge test, the mortality rate of the Fiber 1 group dropped to 10%, the mortality rate of the Fiber 2 group was 20%, and the mortality rates of the LaSota group and the PBS group were 80% and 90% respectively. From the above results, it can be seen that the Newcastle disease virus expressing the Fiber 1 and Fiber 2 of avian adenovirus has a good immune protection effect against FAdV-4, but two immunizations are required to achieve a good protection effect.

[0008] It can be clearly seen that the vaccine prepared from the Fiber 2 recombinant virus has an immune protection rate of only 50% with a single immunization. To achieve a better immune effect, a second immunization is required.

[0009] Prior art 2: CN107475296A discloses a recombinant fowlpox virus transfer vector expressing the fiber2 gene of fowl adenovirus type 4, its construction method and application. The recombinant fowlpox vector vaccine constructed by it can express the fiber 2 protein, with an OD value of 0.75, which is similar to the antibody level of commercially available inactivated vaccines.

[0010] In the prior art, it is very difficult to achieve a high protection rate for immunization against FADV serotype 4 based on the expression of the Fiber-2 gene. Summary of the Invention

[0011] The object of the present invention is to provide two strains of recombinant turkey herpesvirus expressing the spike protein of fowl adenovirus serotype 4, with replication ability consistent with that of the parental virus, and capable of providing good immune protection against FADV-4 virus, meeting the requirements of vaccine candidate strains.

[0012] Meanwhile, the present invention also provides a target gene, uses and vaccines.

[0013] To achieve the above object, the present invention provides the following technical solutions:

[0014] A recombinant turkey herpesvirus expressing the spike protein of fowl adenovirus serotype 4, inserts the target gene between the HVT053 site and the HVT054 site or between the HVT087 site and the HVT088 site of the turkey herpesvirus, and the target gene is the Fiber-2 gene with a promoter and a terminator to obtain a recombinant virus;

[0015] The nucleotide sequence of the Fiber-2 gene is as shown in SEQ ID NO.1.

[0016] In the above recombinant turkey herpesvirus, the nucleotide sequence of the target gene is as shown in SEQ ID NO.2.

[0017] In the above recombinant turkey herpesvirus, the promoter is the mCMV promoter and the terminator is the SV40 terminator.

[0018] Meanwhile, the present invention also discloses a target gene, including the Fiber-2 gene and the promoter and terminator connected to the Fiber-2 gene, and the nucleotide sequence of the Fiber-2 gene is as shown in SEQ ID NO.1.

[0019] In the above target gene, the promoter is the mCMV promoter and the terminator is the SV40 terminator.

[0020] The mCMV promoter sequence refers to SEQ ID NO.6, and the sequence of the SV40 terminator refers to SEQ ID NO.7;

[0021] In addition, the present invention also discloses the use of the recombinant virus as described above in any one for preparing a vaccine for preventing and treating avian adenovirus infection.

[0022] In the above use, the avian adenovirus is avian adenovirus type 4.

[0023] Finally, the present invention also discloses a vaccine containing the recombinant virus as described above in any one.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The present invention successfully constructs HVT-FADV / 053 and HVT-FADV / 087 recombinant viruses, whose replication ability is consistent with that of the parental virus, and can provide good immune protection against FADV-4 type virus, meeting the requirements of vaccine candidate strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Result of amplifying Fiber-2 for FADV SD1504 strain;

[0027] Figure 2 Phylogenetic tree diagram of Hexon gene of FADV SD1504 strain;

[0028] Figure 3 Comparison diagram of gene deletion of FADV SD1504 strain;

[0029] Figure 4A Schematic diagram of the construction of HVT-BAC-Fiber-2 / 053;

[0030] Figure 4B Schematic diagram of the construction of HVT-BAC-Fiber-2 / 087;

[0031] Figure 5A PCR identification result diagram of HVT-BAC-Galk / 053;

[0032] Figure 5B PCR identification result diagram of HVT-BAC-Galk / 087;

[0033] Figure 5C Structural map of pDC315 vector;

[0034] Figure 6A PCR identification result diagram of HVT-BAC-Fiber-2 / 053;

[0035] Figure 6B PCR identification result diagram of HVT-BAC-Fiber-2 / 087;

[0036] Figure 7A Schematic diagram of the construction of HVT-FADV / 053;

[0037] Figure 7B Schematic diagram of the construction of HVT-FADV / 087;

[0038] Figure 8A Amplification diagram after the complete deletion of the BAC sequence in HVT-FADV / 053;

[0039] Figure 8B Amplification diagram after the amplification of the donor sequence in HVT-FADV / 053;

[0040] Figure 8C Amplification diagram after the complete deletion of the BAC sequence in HVT-FADV / 087;

[0041] Figure 8D Amplification diagram after the amplification of the donor sequence in HVT-FADV / 087;

[0042] Figure 9A Result diagram showing that pX458-sgRNA cannot be amplified in HVT-FADV / 053;

[0043] Figure 9B Result diagram showing that pX458-sgRNA cannot be amplified in HVT-FADV / 087;

[0044] Figure 10A Observation result diagram 5 days after HVT-FADV / 053 infects CEF cells;

[0045] Figure 10B Observation result diagram 5 days after HVT-FADV / 087 infects CEF cells;

[0046] Figure 10C Observation result diagram 5 days after HVT infects CEF cells;

[0047] Figure 10D Diagram of CEF cells;

[0048] Figure 11 Growth curves of HVT-FADV / 053, HVT-FADV / 087, and HVT;

[0049] Figure 12A Staining result diagram of cells inoculated with HVT-FADV / 053;

[0050] Figure 12B Staining result diagram of cells inoculated with HVT-FADV / 087;

[0051] Figure 12C The staining result diagram of CEF cells;

[0052] Figure 13 The result diagram of Western-blot staining with chicken Fiber-2 polyclonal antibody as the primary antibody after collecting proteins from LMH cells infected with SD1504 virulent strain and CEF cells infected with recombinant viruses HVT-FADV / 053 and HVT-FADV / 087;

[0053] Figure 14A The PCR detection result of recombinant virus HVT-FADV / 053 after passage;

[0054] Figure 14B The PCR detection result of recombinant virus HVT-FADV / 087 after passage;

[0055] Figure 15A The detection result of Fiber-2 protein expression of recombinant virus HVT-FADV / 053 after passage;

[0056] Figure 15B The detection result of Fiber-2 protein expression of recombinant virus HVT-FADV / 087 after passage;

[0057] Figure 16 The result of serum neutralization titer after immunization with HVT-FADV / 053, HVT-FADV / 087 recombinant viruses and HVT parental virus determined by VN method;

[0058] Figure 17 The result of serum antibody titer after immunization with HVT-FADV / 053, HVT-FADV / 087 recombinant viruses and HVT parental virus determined by indirect ELISA method.

[0059] Figure 18A The survival rate diagram of chickens in each immunized group challenged with SD1504 strain;

[0060] Figure 18B The change diagram of target organ damage of chickens in each immunized group challenged with SD1504 strain;

[0061] Figure 18C The pathological histological change diagram of target organs of chickens in each immunized group after challenge with SD1504 strain;

[0062] Figure 19 The virus content diagram of cloacal swabs of chickens in different immunized groups after challenge;

[0063] Figure 20 The virus content diagram of tissues of chickens in different immunized groups after challenge. Detailed implementation methods

[0064] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0065] Part I Reagents and Consumables

[0066] Commercially purchased: See Table 1

[0067] Self-prepared reagents:

[0068] 0.2 mg / mL biotin: Dissolve 10 mg of biotin in 50 mL of ddH2O. After complete dissolution, filter and sterilize with a 0.22 μm filter membrane and store at 4°C for later use.

[0069] 10 mg / mL L-leucine: Weigh 500 mg of L-leucine and dissolve it in 50 mL of ddH2O. After complete dissolution, filter and sterilize with a 0.22 μm filter membrane and store at 4°C for later use.

[0070] 1 M / L MgSO4: Weigh 12.3 g of MgSO4·7H2O and dissolve it in 50 mL of ddH2O. After complete dissolution, filter and sterilize with a 0.22 μm filter membrane and store at 4°C for later use.

[0071] 20% galactose: Weigh 20 g of galactose and dissolve it in 100 mL of ddH2O. After complete dissolution, autoclave and store at 4°C for later use.

[0072] 20% 2-deoxy-galactose (DOG): Weigh 20 g of DOG and dissolve it in 100 mL of ddH2O. After complete dissolution, autoclave and store at 4°C for later use.

[0073] M9 saline: Weigh 15.13 g of Na2HPO4·12H2O, 3 g of KH2PO4, 1 g of NH4Cl, and 0.5 g of NaCl and dissolve them in 800 mL of ddH2O. After sufficient stirring and dissolution, add ddH2O to make the volume of the solution up to 1 L. Autoclave and store at 4°C for later use.

[0074] 5×M63 culture medium: Weigh 10 g of (NH4)2SO4, 68 g of KH2PO4, and 2.5 mg of FeSO4·7H2O and dissolve them in 800 mL of ddH2O. After sufficient stirring and dissolution, adjust the pH to 7.0 with KOH and make the volume up to 1 L. Autoclave.

[0075] Galactose screening culture plate: Weigh 15 g of agar and dissolve it in 800 mL of ddH2O. After complete dissolution, autoclave at 121 °C for 20 min. After taking it out, add 200 mL of autoclaved 5×M63 culture medium and 1 mL of MgSO4·7H2O, and adjust the volume to 1 L with sterile ddH2O. When the temperature drops to about 50 °C, add 5 mL of 0.2 mg / mL biotin, 4.5 mL of 10 mg / mL L-leucine, 10 mL of 20% galactose, and 500 μL of 50 mg / mL chloramphenicol. After thorough mixing, pour the plate.

[0076] DOG culture plate: Weigh 15 g of agar and dissolve it in 800 mL of ddH2O. After complete dissolution, autoclave at 121 °C for 20 min. After taking it out, add 200 mL of autoclaved 5×M63 culture medium and 1 mL of MgSO4·7H2O, and adjust the volume to 1 L with sterile ddH2O. When the temperature drops to about 50 °C, add 5 mL of 0.2 mg / mL biotin, 4.5 mL of 10 mg / mL L-leucine, 4.5 mL of 10 mg / mL L-isoleucine, 4.5 mL of 10 mg / mL L-valine, 10 mL of 20% DOG, 10 mL of 20% glycerol, and 500 μL of 50 mg / mL chloramphenicol. After thorough mixing, pour the plate.

[0077] MacConkey plate containing galactose: Dissolve 40 g of MacConkey medium in 1 L of ddH2O, boil for 1 min, add 10 mL of 20% galactose, autoclave at 121 °C for 20 min. When the temperature drops to about 50 °C, add chloramphenicol to a final concentration of 30 μg / mL, and pour the plate.

[0078] Washing solution: Take 2000 mL of PBS solution, add 1 mL of Tween-20, mix well and store at room temperature.

[0079] Blocking solution: Dissolve 5 g of skim milk powder in 100 mL of washing solution.

[0080] Stop solution: Measure 30 mL of concentrated sulfuric acid and add it to 240 mL of distilled water to prepare 2 mol / L dilute sulfuric acid.

[0081] Items stored in the laboratory: Fiber 2-coated antigen, LMH cells, HVT-BAC infectious clone, SW102 strain, and peGFP-galK plasmid are stored in the laboratory.

[0082] Experimental animals: SPF chickens and chicken embryos are both purchased from Beijing Boehringer Ingelheim Vetmedica Biotechnology Co., Ltd.

[0083] Table 1 Reagents or consumables

[0084] Reagents or Consumables Company or Brand F12 Culture Factor Medium Maichen Zhongke Maichen (Beijing) Technology Co., Ltd. FBS Thermo Fisher Scientific lnc. DMEM Cell Maintenance Medium Maichen Zhongke Maichen (Beijing) Technology Co., Ltd. Penicillin and Streptomycin Beijing Solarbio Science & Technology Co., Ltd. SOC Beijing Solarbio Science & Technology Co., Ltd. D-(+)Galactose Sigma-Aldrich Brand 2-Deoxy-D-galactose TCI (Shanghai) Chemical Industry Development Co., Ltd. Tissue Cell DNA Extraction Kit Beijing Aidlab Biotechnologies Co., Ltd. Premestar HS DNA polymerase Biolight Biotechnology (Beijing) Co., Ltd. Blunt-Zero Cloning Kit Nanjing Novoprotein Scientific Inc. ELISA Coating Buffer (10×) Beijing Solarbio Science & Technology Co., Ltd. Two-Component TMB Chromogenic Solution Beijing Solarbio Science & Technology Co., Ltd. Tween-20 Beijing Solarbio Science & Technology Co., Ltd. HRP-Labeled Goat Anti-Chicken IgG Beyotime Biotechnology Co., Ltd.

[0085] Table 2 Sequence Information Table

[0086]

[0087]

[0088]

[0089] All primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0090] Isolation and Genetic Evolution Analysis of the Second Part FADV (SD1504)

[0091] The FADV SD1504 strain was isolated and identified from clinically infected broilers in Shandong region in 2015 and stored in the laboratory. After extracting the DNA of the SD1504 strain, using the Fiber-2 open reading frame primers Fiber-2-F / R (Table 2), and amplifying with reference to the PCR reagents and procedures in Table 3, the Fiber-2 gene sequence was obtained ( Figure 1 , M, 5K Maker; 1, FADV SD1504 sample).

[0092] The relevant sequence information of the FADV SD1504 strain can be seen (BankIt2236677Seq1MN091376);

[0093] Table 3 PCR Amplification Reagent Table

[0094] Reagent Name Volume 2×PrimeSTAR GC Buffer 25μl dNTP Mixture 4μl Fiber-2-F 2μl Fiber-2-R 2μl Prime STAR HS DNA Polymerase 1μl <![CDATA[ddH2O]]> 15μl SD1504 DNA 1μl Total 50μl

[0095] The PCR amplification program was: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s, annealing at 60°C for 5 s, extension at 72°C for 1 min 30 s (1 Kbp / min), 30 cycles, 72°C for 10 min, and the expected amplified product size was 1440 bp.

[0096] Genetic evolution analysis: Phylogenetic analysis was performed using the Hexon gene sequence. The genome alignment results showed that the SD1504 strain was closely related to the FADV serotype 4 isolated in China in recent years, and the gene variation was small.

[0097] Using MEGA 7.0 software, a phylogenetic tree of the Hexon gene of 1 isolated strain and 32 reference strains was established by the maximum likelihood method and 1000 bootstrap replications, referring to Figure 2 . The red circle on the phylogenetic tree represents the isolated SD1504 strain.

[0098] Genomic analysis: Some studies have shown that deletions in some tandem repeat regions and some ORFs in the FADV genome are usually associated with increased virulence of FADV serotype 4. Notably, gene deletions were also observed at the 3' end of the SD1504 strain, with a 144 bp deletion in ORF29 of the genome ( Figure 3 ), indicating the high infectivity and pathogenicity of the SD1504 strain to chickens and making it a typical FADV serotype 4 virus.

[0099] Part III Quantification of FADV (SD1504)

[0100] Chicken hepatocarcinoma cells (LMH) can efficiently and stably cultivate high-titer avian adenovirus. Therefore, LMH cells were selected to propagate the SD1504 strain.

[0101] LMH cells were cultured in a 37°C, 5% CO2 incubator for 24 h until 80% confluent, then washed 2 - 3 times with PBS, inoculated with the SD1504 strain, adsorbed at 37°C for 1 h, and then replaced with fresh cell maintenance medium. Cultivation was continued at 37°C for 72 h. The cytopathic effect of the cells was observed under an inverted microscope. When more than 80% of the cells showed cytopathic changes, the complete culture was harvested, frozen and thawed 3 times repeatedly, and the supernatant was collected as the virus solution, which was stored at -80°C for later use.

[0102] The propagated SD1504 strain was serially diluted 10-fold with DMEM / F12 medium containing 2% FBS. Dilutions of 10 -6 , 10 -7 , 10 -8 , 10 -9 were taken, and 0.1 ml of each dilution was inoculated into 3 wells of LMH cells that had grown into a good monolayer on a 96-well cell culture plate; at the same time, 3 wells of normal cells without virus inoculation were set as controls. The cell culture plate was incubated at 37°C and 5% CO2 for 5 days, and the number of wells with cytopathic changes was observed and recorded. The TCID 50 was calculated by the Reed-Muench method. After quantification, the virus content of the propagated SD1504 strain was 2×10 8.5 TCID 50 / 0.1 ml.

[0103] Establishment of an FADV SD1504 animal challenge model:

[0104] To evaluate the immune protection effect of the vaccine, an animal model of SPF chickens infected with the SD1504 strain was constructed for vaccine evaluation.

[0105] First, the median lethal dose of the SD1504 strain for chickens was determined. The quantified SD1504 strain (2×10 8.5 TCID 50 / 0.1 mL) was serially diluted 10-fold with normal saline, and 10 -2 、10 -3 、10 -4 、10 -5 A total of 4 dilution degrees were obtained. Five 49-day-old SPF chickens were inoculated intramuscularly with 1.0 mL per chicken at each dilution degree. After observing for 7 days, the number of dead chickens in each group was recorded, and the LD 50 .

[0106] After determination, the virus content of the SD1504 strain was 10 4.5 LD 50 / 1.0 mL, which was diluted with normal saline to 100 LD 50 / 1.0 mL. Ten 49-day-old SPF chickens were inoculated intramuscularly with 1.0 mL per chicken. All 10 chickens died within 7 days, and the mortality rate was 100%. According to the FADV vaccine test standard, after intramuscularly inoculating 49-day-old SPF chickens with 100 LD 50 / 1.0 mL of the virus solution, the mortality rate should be at least 80% within 7 days, indicating that the SD1504 strain infection and challenge model was successfully constructed.

[0107] Part IV Construction of HVT-FADV Recombinant Live Vector Vaccine

[0108] Previously, using the bacterial artificial chromosome (BAC) technology, an infectious clone of turkey herpesvirus (HVT), HVT-BAC, was constructed (specifically visible in CN109402071A, a recombinant turkey herpesvirus expressing the H9 protein of H9N2 subtype avian influenza virus, paragraphs 92-113 of the specification, I. Construction of recombinant plasmid pBAC-GFP-US and II. Obtaining positive clones of recombinant virus HVT-BAC).

[0109] Using the HVT-BAC infectious clone, a recombinant turkey herpesvirus expressing the fiber protein (Fiber-2) of avian adenovirus serotype 4 was constructed. Briefly, in SW102 bacteria, using the galk screening technology, the Fiber-2 eukaryotic expression cassette containing the mCMV promoter and SV40 terminator was inserted into the HVT-BAC infectious clone at different sites to construct the HVT-BAC-Fiber-2 infectious clone; further using the CRISPR / Cas9-induced homologous recombination technology, in the case of providing an exogenous donor gene donor identical to HVT, the BAC sequence was replaced with the donor gene, thereby obtaining the recombinant turkey herpesvirus HVT-FADV that only inserted the Fiber-2 gene eukaryotic expression cassette. Since the recombinant turkey herpesvirus does not contain GFP (SEQ ID NO.4), the recombinant turkey herpesvirus HVT-FADV can be successfully screened by screening plaques without green fluorescence.

[0110] 4.1 Construction of Recombinant HVT-BAC-Fiber-2 Infectious Clone

[0111] To construct a recombinant turkey herpesvirus expressing fowl adenovirus serotype 4 fiber protein (Fiber-2), the Galk screening technique was used, and with the help of the SW102 genetically engineered bacterium, the Fiber-2 eukaryotic expression cassette was inserted into the HVT-BAC infectious clone in two steps.

[0112] In the first step, using peGFP-galk as a template, it was amplified with primers 053-homo-galk-F / R or 087-homo-galk-F / R containing 50bp homologous arms (Table 2). After purifying the PCR product, it was electrotransformed into the SW102 competent cells containing HVT-BAC for recombination and screened in galactose medium to obtain the SW102 bacteria containing the HVT-BAC-Galk clone.

[0113] In the second step, using the p-T-Fiber-2exp plasmid as a template, it was amplified with primers 053-homo-Fiber-2-F / R and 087-homo-Fiber-2-F / R containing 50bp homologous arms (Table 2). After purifying the corresponding PCR products respectively, they were electrotransformed into the SW102 competent cells containing HVT-BAC-Galk / 053 or HVT-BAC-Galk / 087. After recombination, they were screened in the medium containing 2-deoxy-galactose (DOG) to obtain the SW102 clones containing HVT-BAC-Fiber-2 / 053 ( Figure 4A ), the construction schematic diagram of HVT-BAC-Fiber-2 / 053) and HVT-BAC-Fiber-2 / 087 ( Figure 4B ), the construction schematic diagram of HVT-BAC-Fiber-2 / 087).

[0114] The specific operations are as follows:

[0115] 4.1.1 Preparation of HVT-BAC-galk Infectious Clone:

[0116] Take 500 μl of the SW102 bacterial solution containing the HVT-BAC vector and add it to 10 ml of LBCm+ medium for overnight screening culture. The next day, in a 250 ml conical culture flask with a barrier, take 5 ml of the overnight culture bacterial solution and add it to 150 ml of LBCm+ medium, and culture it in a 32 °C water bath shaker until the OD600 value reaches 0.55 - 0.60.

[0117] Then place the culture solution in a 42 °C water bath shaker for heat shock induction for 15 minutes.

[0118] Cool the induced bacterial solution in an ice-water mixture and transfer it to two 50 ml centrifuge tubes. Centrifuge at 5000 rpm at 4 °C for 5 min. Prepare electrocompetent cells by conventional methods.

[0119] Using peGFP-galk as a template, amplify and purify with primers 053-homo-galk-F / R or 087-homo-galk-F / R (Table 2) containing 50 bp homologous arms to obtain purified 053-homo-galk-F / R or 087-homo-galk-F / R products;

[0120] In this step, the SW102 genetically engineered bacteria and peGFP-galk are described in the following literature: Warming S, Costantino N, Court D L, Jenkins N A, and Copeland N G. Simple and highly efficient BAC recombineering using galK selection. 2005; 33: e36-e36.

[0121] Mix the purified 053-homo-galk-F / R or 087-homo-galk-F / R products with electrocompetent cells and perform electrotransformation. The program is a 0.1 cm electroporation cuvette, and the parameters are 25 μF, 1.75 kV, and 200 Ω.

[0122] After electrotransformation, resuspend with 450 μl of SOC, rejuvenate the bacteria in a shaker at 32 °C for 1-2 h, then centrifuge at 10000 rpm for 1 min, wash the bacteria twice with 1×M9 saline, and culture on a plate of basal medium containing galactose for 3-4 days.

[0123] Use MacConkey plates containing galactose to screen and purify the SW102 bacteria containing the HVT-BAC-Galk / 053 or HVT-BAC-Galk / 087 infectious clones.

[0124] To identify whether the galk gene (SEQ ID NO.5) was inserted into HVT-BAC, PCR identification was performed on the HVT-BAC-Galk / 053 or HVT-BAC-Galk / 087 clones constructed with primers 053-homo-galk-F / R or 087-homo-galk-F / R. The results showed that bands of about 1300 bp could be amplified in both cases, indicating successful construction of HVT-BAC-Galk / 053 ( Figure 5A ) or HVT-BAC-Galk / 087 clones ( Figure 5B ).

[0125] Figure 5A , Detection of galk gene insertion. The samples were amplified using 053-homo-galk-F / R primers. M is the maker; PC is the positive control peGFP-galk; S is HVT-BAC-Galk / 053; NC is the negative control HVT DNA.

[0126] Figure 5B , Detection of galk gene insertion. The samples were amplified using 087-homo-galk-F / R primers. M is the maker; PC is the positive control peGFP-galk; S is HVT-BAC-Galk / 087; NC is the negative control HVT DNA.

[0127] 4.1.2 Preparation of HVT-BAC-Fiber-2 infectious clone:

[0128] First, the Fiber-2 gene was fused with the mCMV promoter and SV40 terminator of the pDC315 vector using the overlap PCR method to prepare a Fiber-2 eukaryotic expression vector. Then, according to the method in the T-vector instruction manual, the Fiber-2 eukaryotic expression cassette was linked to the T-vector to construct p-T-Fiber-2exp. Using the p-T-Fiber-2exp plasmid as a template, the Fiber-2 eukaryotic expression cassette amplification products containing HVT homologous arms at different sites were amplified and purified using 053-homo-Fiber-2-F / R and 087-homo-Fiber-2-F / R primers (Table 2) with 50bp homologous arms, and then inserted between the HVT053 and HVT054 sites and between the HVT087 and HVT088 sites, respectively.

[0129] The structural map of the pDC315 vector can be seen Figure 5C ;

[0130] The pDC315 plasmid is a eukaryotic expression plasmid vector. Inserting the target gene into the multiple cloning site and transfecting cells can enable expression. The pDC315 with the target gene can be identified by the restriction enzyme sites on both sides of the insertion site and the specific restriction enzyme sites within the target gene.

[0131] Figure 5C The information of each component in

[0132] Promoter: mCMV promoter;

[0133] Replicon: pUC ori;

[0134] Terminator: SV40 poly(A) signal;

[0135] Plasmid size: 3913bp;

[0136] Prokaryotic resistance: Ampicillin Amp;

[0137] Ampr: Provides ampicillin resistance (final concentration 100 μg / ml) for plasmid amplification in E. coli (DH5α);

[0138] ori: Replication origin of the plasmid in E. coli;

[0139] Ad: Ad sequence, mainly due to Ad can undergo homologous recombination with the Ad genomic plasmid;

[0140] ITR: Ad inverted terminal repeat sequence, which is the packaging recognition signal of the Ad genome;

[0141] mCMV: Immediate early promoter from murine CMV virus;

[0142] SV40 polyA: mRNA polyadenylation signal from SV40 virus T antigen;

[0143] loxP: Recombination recognition sequence of eukaryotic recombinase (Cre).

[0144] To obtain the infectious clones of HVT-BAC-Fiber-2 / 053 and HVT-BAC-Fiber-2 / 087, according to the second step of galk screening, electrotransformation and recombination were carried out.

[0145] The specific steps are as follows:

[0146] 500 μl of SW102 bacterial solution containing the infectious clone of HVT-BAC-Galk / 053 or HVT-BAC-Galk / 087 was added to 10 ml of LBCm+ medium and cultured overnight. The next day, in a 250 ml conical culture flask with a barrier, 5 ml of the overnight cultured bacterial solution was taken and added to 150 ml of LBCm+ medium, and cultured in a water bath shaker at 32 °C until the OD600 value reached 0.55 - 0.60.

[0147] Then transfer the bacterial solution to a water bath shaker at 42 °C for heat shock for 15 minutes.

[0148] The induced bacterial solution was cooled in an ice-water mixture and transferred to two 50 ml centrifuge tubes, centrifuged at 5000 rpm at 4 °C for 5 min, and electrocompetent cells were prepared by the conventional method.

[0149] Using the p-T-Fiber-2exp plasmid as a template, amplify and purify with the 053-homo-Fiber-2-F / R and 087-homo-Fiber-2-F / R primers (Table 2) containing 50bp homologous arms to obtain the purified 053-homo Fiber-2-F / R or 087-homo Fiber-2-F / R amplification products;

[0150] After mixing the purified 053-homo Fiber-2-F / R or 087-homo Fiber-2-F / R amplification products with electrotransformation-competent cells, perform electrotransformation using a 0.1 cm cuvette with parameters of 25 μF, 1.75 KV, and 200 Ω.

[0151] After electrotransformation, resuspend with 450 μl of SOC, rejuvenate the bacteria in a shaker at 32 °C for 1 - 2 h, then centrifuge at 10000 rpm for 1 min, wash twice with 1×M9 saline, and spread on a basal medium plate containing deoxygalactose (DOG) and culture for 4 days. Finally, obtain SW102 bacteria containing HVT-BAC-Fiber-2 / 053 and HVT-BAC-Fiber-2 / 087.

[0152] To identify whether the galk gene was completely deleted, perform PCR identification on the HVT-BAC-Fiber-2 / 053 or HVT-BAC-Fiber-2 / 087 clones constructed using the 053-homo-galk-F / R and 087-homo-galk-F / R primer pairs. As a result, no corresponding bands were amplified in HVT-BAC-Fiber-2 / 053 ( Figure 6A ) and HVT-BAC-Fiber-2 / 087 ( Figure 6B ), indicating that the galk sequence has been completely deleted.

[0153] Figure 6A , Detection of galk gene deletion. Amplify the sample using the 053-homo-galk-F / R primer. M, 2K maker; 1, HVT-BAC-Fiber-2 / 053; PC, positive control peGFP-galk; NC, negative control HVT DNA.

[0154] Figure 6B , Detection of galk gene deletion. Amplify the sample using the 087-homo-galk-F / R primer. M, 2K maker; 1, HVT-BAC-Fiber-2 / 087; PC, positive control peGFP-galk; NC, negative control HVT DNA.

[0155] 4.2 Preparation of recombinant live vector vaccine HVT-FADV

[0156] To delete the BAC sequence, with the aid of the CRISPR / Cas9 technology, first, sgRNA primers (sgRNA-F / R, Table 2) were designed and synthesized, and the sgRNA was linked to pX458 to construct the CRISPR plasmid pX458-sgRNA containing the sgRNA. The operation steps refer to the patent CN109402071A of the present inventor, a recombinant turkey herpesvirus expressing the H9 protein of H9N2 subtype avian influenza virus, which has been described in detail in the specification, see paragraphs 138-145 of the specification.

[0157] To completely delete the BAC sequence and make it identical to the parental virus, a donor gene (donor-F / R, Table 2) identical to the HVT parental virus sequence was used. The DNA double-strand breaks formed by CRISPR / Cas9 cleavage of HVT-BAC-Fiber-2 / 053 and HVT-BAC-Fiber-2 / 087 were utilized to induce homologous recombination repair, enabling the donor gene to replace the BAC sequence, thereby deleting the BAC sequence and obtaining the recombinant viruses HVT-FADV / 053 ( Figure 7A ) and HVT-FADV / 087 ( Figure 7B ). Figure 7A Schematic diagram for the construction of HVT-FADV / 053; Figure 7B Schematic diagram for the construction of HVT-FADV / 087.

[0158] The specific operation is as follows:

[0159] The constructed pX458-sgRNA, donor gene, and HVT-BAC-Fiber-2 / 053 plasmid or HVT-BAC-Fiber-2 / 087 plasmid were co-transfected into 6-well CEF cells that had grown to 90% confluence in advance. After transfection, they were passaged into new CEF cells for screening at 6-7 days, and plaques without green fluorescence were screened to obtain HVT-FADV / 053 and HVT-FADV / 087.

[0160] The sequence information of the donor gene is: referring to SEQ ID NO.3, and its specific preparation process is: using the genomic DNA of HVT as a template, PCR amplification was performed with the primer pair composed of donor-F and donor-R to obtain a DNA fragment (139462nt - 141036nt of the genomic DNA of HVT);

[0161] To identify whether the BAC sequence was completely deleted, PCR identification of the recombinant virus DNA of HVT-FADV / 053 and HVT-FADV / 087 was performed using GFP F / R and donor F / R primers (Table 2). If the BAC sequence was completely deleted, the 720 bp GFP band ( Figure 8A , 8C) could not be amplified, and the 1575 bp donor band ( Figure 8B , 8D) could be amplified. The results were consistent with the expectations, indicating that the BAC sequence had been completely deleted.

[0162] Figure 8A : M, 2K maker; 1, HVT-BAC-Fiber-2 / 053 containing the reporter gene; 2, HVT-FADV / 053 with the reporter gene deleted; PC, positive control pcDNA-GFP; NC, negative control HVT.

[0163] Figure 8C : M, 2K maker; 1, HVT-BAC-Fiber-2 / 087 containing the reporter gene; 2, HVT-FADV / 087 with the reporter gene deleted; PC, positive control pcDNA-GFP; NC, negative control HVT.

[0164] The samples were amplified using donor-F / R primers.

[0165] Figure 8B : M, 2K maker; 1, HVT-FADV / 053 in which the reporter gene was deleted by replacing it with the donor gene; PC, positive control HVT; NC negative control CEF DNA.

[0166] Figure 8D : M, 2K maker; 1, HVT-FADV / 087 in which the reporter gene was deleted by replacing it with the donor gene; PC, positive control HVT; NC negative control CEF DNA.

[0167] To detect whether pX458-sgRNA remained in the recombinant virus, primer CAG-R (Table 2) was designed, and the recombinant virus DNA of HVT-FADV / 053 and HVT-FADV / 087 was extracted. Then, using sgRNA-F and CAG-R as primers and the pX458-sgRNA plasmid as a positive control, PCR identification was performed. If the plasmid pX458-sgRNA remained in the CEF cells or was recombined into the recombinant virus genome, a 750 bp target fragment could be amplified. The results showed that the 750 bp target fragment could not be amplified from the extracted recombinant virus DNA, so the pX458-sgRNA vector was successfully removed ( Figure 9A andFigure 9B )。

[0168] Figure 9A : M, 2K maker; 1, HVT-FADV / 053 virus DNA; PC, pX458-sgRNA plasmid; NC, water negative control.

[0169] Figure 9B : M, 2K maker; 1, HVT-FADV / 087 virus DNA; PC, pX458-sgRNA plasmid; NC, water negative control.

[0170] Part V Identification of Biological Characteristics of HVT-FADV Recombinant Virus

[0171] 5.1 Plaque Morphology of HVT-FADV Recombinant Virus

[0172] Infect CEF cells with HVT-FADV / 053, HVT-FADV / 087 viruses and HVT parental virus respectively, and observe the plaque morphology using an inverted microscope 5 days after infection.

[0173] Reference Figures 10A to 10D , the results show that the plaque morphology and size of the recombinant virus HVT-FADV virus are similar to those of the parental virus HVT; there is not much difference in the plaque morphology between HVT-FADV / 053, HVT-FADV / 087 viruses and the parental virus.

[0174] Figure 10A is the observation result graph of CEF cells 5 days after infection with HVT-FADV / 053;

[0175] Figure 10B is the observation result graph of CEF cells 5 days after infection with HVT-FADV / 087;

[0176] Figure 10C is the observation result graph of CEF cells 5 days after infection with HVT;

[0177] Figure 10D is the graph of CEF cells;

[0178] Magnification is 100 times.

[0179] 5.2 Growth Curve of HVT-FADV Recombinant Virus

[0180] Prepare CEF cells and seed them in a 6-well cell plate. The next day, inoculate each well of CEF cells with 100 PFU doses of HVT-FADV / 053, HVT-FADV / 087 and HVT parental virus respectively; collect 3 wells at 24, 48, 72, 96, 120 h after inoculation and quantify the virus using the 2-fold serial dilution method, and finally draw the growth curve. Reference Figure 11The results showed that there was no difference in the in vitro growth and replication ability of the recombinant viruses HVT-FADV / 053, HVT-FADV / 087 and the parent virus HVT.

[0181] Figure 11 These are the growth curves of HVT-FADV / 053, HVT-FADV / 087, and HVT.

[0182] 5.3 IFA detection of the expression of the exogenous gene Fiber-2 of HVT-FADV recombinant virus

[0183] CEF cells were prepared and inoculated with recombinant viruses HVT-FADV / 053 and HVT-FADV / 087. After 72 h of infection, the prepared chicken Fiber-2 polyclonal antibody was used as the primary antibody, and FITC-labeled goat anti-chicken IgG secondary antibody was used for IFA staining ( Figure 12A , Figure 12B and Figure 12C ).

[0184] The results are as follows: The cells inoculated with HVT-FADV / 053 and HVT-FADV / 087 showed obvious expression of Fiber-2 protein.

[0185] Figure 12A This is a staining result of cells inoculated with HVT-FADV / 053;

[0186] Figure 12B This is the staining result of cells inoculated with HVT-FADV / 087;

[0187] Figure 12C This is the staining result of CEF cells.

[0188] Magnification: 100x.

[0189] 5.4 Western-blot detection of the expression of the exogenous gene Fiber-2 of the HVT-FADV recombinant virus

[0190] After 72 h of infection of cells with recombinant viruses HVT-FADV / 053 and HVT-FADV / 087, proteins in 6-well plates were collected and Western-blot identification was performed using chicken Fiber-2 polyclonal antibody as the primary antibody ( Figure 13 ), the results showed that the Fiber-2 band of about 70KD could be detected.

[0191] Figure 13Results of Western-blot staining with chicken Fiber-2 polyclonal antibody as the primary antibody after collecting proteins from LMH cells infected with SD1504 virus and CEF cells infected with recombinant viruses HVT-FADV / 053 and HVT-FADV / 087;

[0192] Among them, 1 is LMH cells infected with SD1504 virus; 2 is HVT-FADV / 053; 3 is HVT-FADV / 087; NC is HVT virus;

[0193] 5.5 Genetic stability of HVT-FADV recombinant virus

[0194] To detect the genetic stability of HVT-FADV virus, 100 PFU of HVT-FADV / 053 and HVT-FADV / 087 recombinant viruses were used to infect CEF cells. After 1 h of infection, the cell supernatant was discarded, the cells were washed twice with PBS, and the DMEM cell maintenance medium containing 1% FBS was replaced. After culturing for 3 - 4 days, the cells were digested, and a certain amount of virus was taken and inoculated onto new CEF cells. This operation was repeated, and the virus was passaged continuously for 20 generations, and the virus was collected every 5 generations. After extracting DNA from the collected virus, PCR identification was performed using Fiber-2-F / R primers (Table 2) ( Figure 14A and Figure 14B ), and Western-blot detection was used to detect the expression of virus Fiber-2 protein ( Figure 15A and Figure 15B ).

[0195] Figure 14A PCR detection results of recombinant virus HVT-FADV / 053 after passage;

[0196] Figure 14B PCR detection results of recombinant virus HVT-FADV / 087 after passage;

[0197] Figure 14A : M, marker; P5, P10, P15, P20 represent the DNA of HVT-FADV / 053 virus extracted from the 5th, 10th, 15th, and 20th passages of the virus respectively; PC, DNA of SD1504 virus as a positive control; NC, DNA extracted from CEF cells as a negative control.

[0198] Figure 14B : M, marker; P5, P10, P15, P20 represent the DNA of HVT-FADV / 087 virus extracted from the 5th, 10th, 15th, and 20th passages of the virus respectively; PC, DNA of SD1504 virus as a positive control; NC, DNA extracted from CEF cells as a negative control.

[0199] Figure 15A It is the detection result of the expression of Fiber-2 protein after the passage of the recombinant virus HVT-FADV / 053;

[0200] Figure 15B It is the detection result of the expression of Fiber-2 protein after the passage of the recombinant virus HVT-FADV / 087;

[0201] Figure A: Lanes 1, 2, 3, and 4 represent the detection of cell lysates after the 5th, 10th, 15th, and 20th passages of HVT-FADV / 053 virus infecting CEF; Lane 5 is the detection of cell lysates after SD1504 virus infecting LMH, as a positive control; Lane NC is the detection of cell lysates after HVT infecting CEF, as a negative control.

[0202] Figure B: Lanes 1, 2, 3, and 4 represent the detection of cell lysates after the 5th, 10th, 15th, and 20th passages of HVT-FADV / 087 virus infecting CEF; Lane 5 is the detection of cell lysates after SD1504 virus infecting LMH, as a positive control; Lane NC is the detection of cell lysates after HVT infecting CEF, as a negative control;

[0203] The results show that the Fiber-2 gene of HVT-FADV can be stably inherited.

[0204] Part VI Immunoprotective Evaluation of HVT-FADV Recombinant Virus

[0205] 6.1 Determination of Serum Antibody Neutralization Titer

[0206] Take the sera of chickens immunized with HVT-FADV / 053, HVT-FADV / 087 recombinant viruses and HVT parental virus at different weeks, make 2-fold serial dilutions with cell maintenance medium containing 2% FBS, and mix them equally with SD1504 virus solution (containing 200 TCID 50 / 0.1 ml), incubate at 37°C for 60 minutes, inoculate 3 wells of a 96-well cell culture plate containing monolayer LMH cells at each dilution, 100 μl / well; set 3 wells of virus control, inoculate 100 μl of an equal mixture of virus solution and cell maintenance medium treated under the same conditions per well, incubate at 37°C, 5% CO2 for 5 days, and record the cytopathic effect. Calculate the serum neutralization titer according to Reed-Muench.

[0207] The results are referred to Figure 16 , Figure 16 It is the result of the serum neutralization titer determined by the VN method for HVT-FADV / 053, HVT-FADV / 087 recombinant viruses and HVT parental virus after immunization.

[0208] 6.2 Determination of Serum Antibody Titer

[0209] Collect the sera of chickens immunized with HVT-FADV / 053, HVT-FADV / 087 recombinant viruses and HVT parental virus at different weeks after immunization, and determine them by indirect ELISA method. The specific steps are as follows:

[0210] Indirect ELISA operation steps:

[0211] (1) Dilute Fiber 2 protein with 1×ELISA coating buffer. Add 100 μl of the diluted Fiber 2 protein (2 μg / ml) to each well of the ELISA microtiter plate and coat it overnight at 4°C.

[0212] (2) Add 300 μl of PBST to each well and wash the reaction plate 3 times, 5 minutes each time.

[0213] (3) Add 5% skim milk to the microtiter plate, 300 μl per well, and incubate it in a 37°C incubator for 2 hours for blocking.

[0214] (4) Add 300 μl of PBST to each well and wash the reaction plate 3 times, 5 minutes each time.

[0215] (5) Dilute the serum sample to be tested with the blocking solution (diluted 1:400 times). Add 100 μl of the diluted serum to each well of the ELISA microtiter plate and incubate it in a 37°C incubator for 2 hours

[0216] (6) Add 300 μl of PBST to each well and wash the reaction plate 3 times, 5 minutes each time.

[0217] (7) Dilute the enzyme-labeled secondary antibody with the blocking solution. Add 100 μl of the diluted enzyme-labeled secondary antibody (diluted 1:8000 times) to each well of the ELISA microtiter plate and incubate it in a 37°C incubator for 2 hours

[0218] (8) Add 300 μl of PBST to each well and wash the reaction plate 3 times, 5 minutes each time.

[0219] (9) Mix the chromogenic solutions A and B. Add 100 μl of the mixed chromogenic solution to each well. Incubate the microtiter plate in a 37°C incubator for 10 minutes for color development.

[0220] (10) Add 50 μl of 2 M sulfuric acid to each well to terminate the color reaction. Place the microtiter plate in an ELISA reader and measure the OD value (OD450) at a wavelength of 450 nm.

[0221] (11) The critical value of OD450 nm for positive and negative is 0.20. If the OD450 value ≥ 0.20, it is determined as positive; if the OD450 value < 0.26, it is determined as negative.

[0222] Result referenceFigure 17 , Figure 17 Results of indirect ELISA for detecting serum antibody titers after immunization with HVT-FADV / 053, HVT-FADV / 087 recombinant viruses and HVT parental virus.

[0223] 6.3 Evaluation of the protective effect against challenge with FADV-4 strain SD1504

[0224] At 4 weeks after immunization, i.e., at 28 days of age, chickens were challenged with 100 LD 50 dose with the FADV-4 SD1504 strain. After challenge, the mental state and mortality of the chickens were observed. Blood was collected weekly for one week after challenge to isolate serum, and at the same time, the chickens were dissected to observe tissue and organ lesions and histological changes. The results showed that all chickens in the HVT challenge control group died within 3 days after challenge, and the mortality rate reached 100% ( Figure 18A ).

[0225] Chickens immunized with HVT-FADV / 053 and HVT-FADV / 087 recombinant viruses were all protected from infection with the SD1504 strain, and the protection rate was 100%. The dissection results showed that, macroscopically, chickens in the HVT challenge control group had obvious enlargement, yellowing, and bleeding of the liver and swelling and bleeding of the kidneys, while all tissues and organs of chickens immunized with HVT-FADV / 053 and HVT-FADV / 087 recombinant viruses were normal ( Figure 18B ); the histological results were consistent with the macroscopic observations. The HVT challenge control group had obvious necrosis of cardiomyocytes, degeneration and necrosis of hepatocytes, hemorrhagic basophilic inclusion bodies, and degeneration and necrosis of renal tubular epithelial cells; there were no obvious cytopathic effects after immunization with the recombinant virus ( Figure 18C ).

[0226] Figure 18A Survival rate of chickens in each immunized group challenged with the SD1504 strain;

[0227] Figure 18B Changes in target organ damage of chickens in each immunized group challenged with the SD1504 strain;

[0228] Figure 18C Histological changes in target organs of chickens in each immunized group after challenge with the SD1504 strain.

[0229] 6.4 Detection of virus shedding amount

[0230] Cloacal swabs were collected from chickens in the HVT control group, the HVT-FADV / 053 immunized group, and the HVT-FADV / 087 immunized group at 3, 5, and 7 days after virus challenge. After adding the sampling solution containing penicillin and streptomycin, the samples were repeatedly frozen and thawed three times. Subsequently, the supernatant was serially diluted and inoculated into LMH cells. The cells were cultured at 37°C and 5% CO2 for 5 days, and the cytopathic effect was recorded. The viral loads in various tissues and organs were determined by the Reed-Muench method.

[0231] Referring to Figure 18, compared with the virus-challenged control group, the viral loads in the cloacal swabs of chickens immunized with the HVT-FADV / 053 and HVT-FADV / 087 recombinant viruses decreased significantly, indicating that the HVT-FADV / 053 and HVT-FADV / 087 recombinant viruses effectively blocked the infection and virus excretion of SD1504 virus. All chickens in the HVT virus-challenged control group died on the third day after virus challenge. The detection results of the viral loads in their cloacal swabs showed that they all had high viral contents.

[0232] Figure 19 It is a graph of the viral contents in the cloacal swabs of chickens in different immunized groups after virus challenge.

[0233] 6.5 Viral loads in various organs

[0234] Hearts, livers, spleens, kidneys, ceca, and bursae of Fabricius from chickens in the HVT control group, the HVT-FADV / 053 immunized group, and the HVT-FADV / 087 immunized group were collected at 3, 5, and 7 days after virus challenge for viral load detection. The tissues and organs were ground into a homogenized state, and after three cycles of repeated freezing and thawing, the sampling solution containing penicillin and streptomycin was added. Subsequently, the supernatant was obtained by centrifugation, serially diluted, and inoculated into LMH cells. The cells were cultured at 37°C and 5% CO2 for 5 days, and the cytopathic effect was recorded. The viral loads in various tissues and organs were determined by the Reed-Muench method.

[0235] Reference Figure 20 , compared with the virus-challenged control group, the viral loads in various tissues and organs of chickens immunized with the HVT-FADV / 053 and HVT-FADV / 087 recombinant viruses decreased significantly, indicating that the HVT-FADV / 053 and HVT-FADV / 087 recombinant viruses effectively exerted their immune effects and inhibited the replication of SD1504 virus in target organs. All chickens in the HVT virus-challenged control group died on the third day after virus challenge. The detection results of the viral loads in their respective target organs showed that they all had high viral contents.

[0236] Figure 20 It is a graph of the tissue viral contents of chickens in different immunized groups after virus challenge.

[0237] Result analysis

[0238] 1. The replication ability of the recombinant virus of the present invention is the same as that of the parental virus;

[0239] 2. It overcomes the defect of the low protection rate of the traditional Fiber-2 recombinant virus. After immunization with the recombinant virus of the present invention, the chickens have no death and the protection rate is 100%.

Claims

1. A recombinant turkey herpesvirus expressing fowl adenovirus serotype 4 fiber protein, characterized in that, Insert the target gene between the HVT053 locus and the HVT054 locus or between the HVT087 locus and the HVT088 locus of the turkey herpesvirus, and the target gene is the Fiber-2 gene with a promoter and a terminator to obtain a recombinant virus; The nucleotide sequence of the Fiber-2 gene is shown in SEQ ID NO.

1.

2. The recombinant turkey herpesvirus according to claim 1, wherein, The nucleotide sequence of the target gene is shown in SEQ ID NO.

2.

3. The recombinant turkey herpesvirus according to claim 1, characterized in that, The promoter is the mCMV promoter, and the terminator is the SV40 terminator.

4. Use of the recombinant virus according to any one of claims 1-3 for preparing a vaccine for preventing and treating avian adenovirus infection; the avian adenovirus is avian adenovirus type 4.

5. A vaccine, characterized in that, Containing the recombinant virus according to any one of claims 1-3.

Images