Recombinant avian pox virus and construction method thereof

Abstract

The invention provides a recombinant avian pox virus and a construction method thereof. According to the invention, the FPV-ILTV is constructed, and the vaccine prepared from the recombinant avian pox virus can generate a relatively high immune protection level and has an immune protection effect on chickens, especially chicks. The live vector vaccine is prepared by mixing the two recombinant avian pox viruses, namely, the FPV-AEV and the FPV-ILTV, according to a proportion, and the live vector vaccine can be used for immunizing an experimental subject and inducing immune response generated by an organism, and can be used as the live vector vaccine for preventing the AEV and the ILTV.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering vaccines, and in particular relates to a recombinant fowlpox virus and a construction method thereof. Background Art

[0002] Avian encephalomyelitis (AE) is an infectious disease caused by the avian encephalomyelitis virus (AEV) that attacks the central nervous system of chicks and laying hens, resulting in a non-suppurative encephalitis. Infectious laryngotracheitis (ILT) is an acute respiratory infection caused by the infectious laryngotracheitis virus (ILTV). Both AEV and ILTV infections can cause chick losses and reduced egg production in hens, posing a significant threat to the poultry industry and representing a major global threat to poultry production. Currently, there are no effective treatments for AEV and ILTV, and prevention relies primarily on vaccination. However, the production of MLV vaccines is complex, ineffective, and carries the risk of reversion to virulence. Some MLV vaccines can also cause disease in chickens and cannot distinguish between field and vaccine strains. Therefore, in order to reduce the impact of factors such as vaccination stress on the poultry industry, the research and development of combined vaccines has become a development trend in the animal vaccine industry.

[0003] Fowlpox virus (FPV) has the largest known animal viral genome and is a widely used tool in the development of genetically engineered live vector vaccines. Currently, protective antigen genes from avian pathogens have been expressed within FPV or vector systems, resulting in recombinant FPV vaccines. These vaccines, for example, express the F and HN genes of Newcastle disease virus and the HA gene of the H5 subtype avian influenza virus, all of which have demonstrated modest immune responses. However, there are no reports of expressing genes from avian infectious laryngotracheitis virus within recombinant FPV or vector systems, nor are there reports of a combined recombinant FPV vaccine for AEV and ILTV. Summary of the Invention

[0004] The object of the present invention is to provide a recombinant fowlpox virus.

[0005] The purpose of the present invention is to provide a method for constructing a recombinant fowlpox virus.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The recombinant fowlpox virus is prepared by cloning the P1 and 3C genes of avian encephalomyelitis virus AEV and the UL32 and gB genes of avian infectious laryngotracheitis virus ILTV into a fowlpox virus FPV vector, thereby packaging the recombinant fowlpox viruses FPV-AEV and FPV-ILTV.

[0007] A recombinant fowlpox virus live vector vaccine is prepared by mixing the recombinant fowlpox viruses FPV-AEV and FPV-ILTV in a certain proportion.

[0008] Preferably, the method for constructing the recombinant fowlpox virus FPV-ILTV comprises the following steps: S1. cDNA fragments of the UL32 and gB genes of avian infectious laryngotracheitis virus (ILTV) were obtained by PCR amplification; S2. The amplified UL32 and gB gene fragments were fused to obtain ILTV-UL32-gB; S3. The fowlpox virus early and late promoters were connected to ILTV UL32-gB to obtain an ILTV UL32-gB expression cassette; S4. The ILTV UL32-gB expression cassette was inserted into the pBlue-FPV-244 vector containing the eGFP expression cassette by in-fusion technology to generate the recombinant plasmid pB-FPV-ILTV; S5. The pB-FPV-ILTV plasmid was transfected into CEF cells. After screening for positive clones, the selection gene was deleted using the Cre enzyme to obtain the recombinant fowlpox virus FPV-ILTV. Preferably, in step S1, the avian infectious laryngotracheitis virus UL32 and gB genes are reversely linked via a promoter.

[0009] Preferably, the fowlpox virus early and late promoter in step S3 is PE / L.

[0010] Preferably, the fowlpox virus early promoter in step S3 is P7.5.

[0011] Preferably, the nucleotide sequence of the ILTV UL32-gB expression cassette in step S3 is shown as SEQ ID NO.1.

[0012] Preferably, the eGFP expression cassette described in step S4 contains LOXP sequences on both sides.

[0013] Application of recombinant fowlpox virus in the preparation of avian encephalomyelitis virus and avian infectious laryngotracheitis virus vaccines.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention inserts the UL32 and gB gene expression frameworks of ILTV into the genome of the FPV attenuated live vaccine strain, constructs a recombinant virus expressing the ILTV UL32 protein and gB protein, and improves its immunogenicity.

[0015] The present invention adopts the fowlpox virus gene early and late promoter PE / L and P7.5 promoter to respectively start the transcription of ILTV UL32 protein and gB gene, so that they can be efficiently expressed in fowlpox virus.

[0016] The use of recombinant fowlpox virus vectors to prepare AEV and ILTV bivalent vaccine for immunization has successfully established a system method for the administration of a bivalent genetically engineered subunit vaccine of AEV and ILTV based on fowlpox virus. No systemic adverse reactions caused by the vaccine occurred after immunization of animals, and the vaccine has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings: Figure 1 Schematic diagram of the construction of recombinant plasmid; Figure 2 Schematic diagram of recombinant virus rescue; Figure 3 The identification results of PCR amplification are shown in the figure; (1) is the amplification result of UL32 F and UL32 R primers, M: Maker; 1-4: 3rd, 5th, 11th, and 15th generation recombinant virus FPV-ILTV; 5: FPV virus control; 6: positive plasmid control; 7: ddH2O control; (2) is the amplification result of gB F and gB R primers, M: Maker; 8-11: 3rd, 5th, 11th, and 15th generation recombinant virus FPV-ILTV; 12: FPV virus control; 13: positive plasmid control; 14: ddH2O control; Figure 4 The indirect immunofluorescence identification results of the recombinant virus are shown in Figure 1, where A is the recombinant fowlpox virus FPV-ILTV; B is the parent FPV control; Figure 5 This is a diagram showing the results of the immune protection test. DETAILED DESCRIPTION

[0018] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0019] Unless otherwise specified, the test methods used in the following experimental examples are conventional methods.

[0020] Example 1. Construction of recombinant fowlpox virus FPV-ILTV 1 Experimental Materials 1.1 Strains, strains, and plasmids The attenuated strain of fowlpox virus was purchased from Ruipu (Baoding) Biopharmaceutical Co., Ltd., the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the enzymes, carriers and other reagents were purchased from Novozymes Biotechnology Co., Ltd.

[0021] Escherichia coli DH5α competent cells were purchased from Novazonics Biotechnology Co., Ltd.; pBlue-FPV-244 plasmid was preserved by the Biological Products Research Institute of Tianjin Ringpu Biotechnology Co., Ltd.; 9-11 day old SPF chicken embryos were purchased from Beijing Boehringer; primary chicken embryo fibroblasts were prepared from 9-11 day old chicken embryo tissue.

[0022] Experimental methods 2.1 Construction process of recombinant fowlpox virus 2.1.1 Construction of recombinant fowlpox virus expression plasmid The genomic RNA of ILTV WG strain of avian infectious laryngotracheitis virus was extracted, and the cDNA obtained by reverse transcription was used as a template and UL32-F and UL32-R, gB-F and gB-R were used as primers to PCR amplify the UL32 and gB fragments respectively. At the same time, the PE / L early and late promoters of fowlpox virus were introduced into the 5' end of the UL32 fragment by the synthetic long primer UL32-R, and the P7.5 early and late promoters of fowlpox virus were introduced into the 5' end of the gB fragment by the synthetic long primer gB-F. Then, by in-fusion homologous recombination, the ILTV UL32-gB was inserted into the pBlue-FPV-244 vector containing the screening gene eGFP gene that had been digested with Srf I to obtain the expression cassette for expressing the ILTV UL32 and gB genes. The constructed intermediate plasmid was named pB-FPV-ILTV, and the gene sequence of the UL32 and gB gene expression cassettes of avian infectious bronchitis virus was shown in SEQ ID NO. 1. The construction schematic diagram is shown in FIG. Figure 1 The primer sequences are shown in Table 1.

[0023] Table 1: Primers used for FPV-ILTV-eGFP construction

[0024] 2.1.2 Transfection and purification of recombinant fowlpox virus The specific method for constructing the recombinant fowlpox virus is as follows: Transfection was performed according to the instructions for Lipofectamine™ 3000 transfection reagent. CEF cells were cultured in six-well plates until they formed a monolayer. The cells were infected with a 0.1 MOI of a weak FPV strain and incubated at 37°C for 3–4 hours. CEF cells already infected with fowlpox virus (FPV) were then transfected with the recombinant plasmid FPV-ILTV-eGFP at a transfection dose of 2 μg. Homologous recombination between the recombinant FPV-ILTV-eGFP plasmid and FPV genomic DNA occurred via homologous arms, inserting the ILTV UL32 and gB genes into the fowlpox virus genome, generating the recombinant virus. This recombinant fowlpox virus contained the eGFP gene. Green fluorescence was observed using an inverted fluorescence microscope 72 hours after transfection, indicating that the FPV-ILTV-eGFP plasmid was expressed in the CEF cells. Plaques with green fluorescence were selected and screened and purified multiple times to obtain relatively pure recombinant virus until all plaques produced by the purified recombinant virus exhibited green fluorescence under an inverted fluorescence microscope. The resulting recombinant virus was named FPV-ILTV-eGFP. To eliminate the eGFP gene, FPV-ILTV-eGFP was inoculated into CEF cells for expansion. The recombinant virus FPV-ILTV-eGFP was then inoculated into CEF cells at an MOI of 0.1. 24 hours later, the cells were transfected with the Cre expression plasmid pCDNA3.1-cre. Transfection was performed according to the instructions for Lipofectamine™ 3000 transfection reagent, with a transfection dose of 2 μg. Because eGFP contains LOXP sites at both ends, homologous recombination occurs under the action of the Cre enzyme. 72 hours after transfection, observe under a fluorescence microscope, mark the plaques without green fluorescence, then digest them, continue to inoculate chicken embryo fibroblasts, observe under a fluorescence microscope, and continue to pick the plaques without green fluorescence until all fields of view are free of fluorescence. The recombinant virus obtained in this way is a recombinant virus without fluorescence, and the recombinant virus is named FPV-ILTV.

[0025] 2.2 Identification of recombinant viruses 2.2.1 Identification of recombinant fowlpox virus The purified recombinant FPV-ILTV virus was passaged in vitro for 20 consecutive generations. Changes in the UL32 and gB genes were examined by PCR every few generations. Viral DNA was extracted and used as a template for PCR amplification using primers UL32 F and UL32 R, and gB F and gB R (see Table 2). Non-recombinant fowlpox virus and ddH2O were also used as controls. PCR amplification of the recombinant fowlpox virus yielded a 1.7 kb UL32 gene fragment and a 2.7 kb gB fragment, indicating successful recombination between the FPV-ILTV-eGFP plasmid and fowlpox virus (see Table 2). Figure 3 ).

[0026] Table 2 Identification primers

[0027] 2.2.2 Results of indirect immunofluorescence identification of recombinant viruses CEF cells were infected with non-recombinant fowlpox virus and purified recombinant fowlpox virus at an MOI of 0.01, respectively. When lesions appeared on the infected CEF, the expression of the target gene was detected by indirect immunofluorescence assay using positive serum of avian infectious bronchitis virus as the primary antibody and Alexa Fluor-488-labeled goat anti-chicken fluorescent secondary antibody. The lesion area of ​​CEF cells inoculated with recombinant fowlpox virus emitted bright green fluorescence, while no green fluorescence was detected in CEF cells inoculated with non-recombinant fowlpox virus, indicating that the target gene was well expressed in the recombinant fowlpox virus (see Figure 4 ).

[0028] The above results indicate that the recombinant virus FPV-ILTV can be stably propagated in chicken embryo fibroblasts.

[0029] Example 2. Evaluation of immune protection of recombinant fowlpox virus vector vaccine FPV-ILTV Fifty one-day-old SPF chickens were randomly divided into five groups: FPV-ILTV, commercial ILTV vaccine, FPV-gB, FPV, and a blank control group. At day one, the FPV-ILTV, commercial ILTV vaccine, FPV-gB, and FPV groups were subcutaneously inoculated with 10,000 PFU / bird of the recombinant FPV-ILTV, commercial ILTV vaccine, FPV-gB control vaccine expressing a single gB gene of ILTV, and control FPV vaccine, respectively. The blank control group was inoculated with only the vaccine diluent. Twenty-one days after immunization, each group was challenged with a virulent ILTV WG strain via the laryngeal inoculation route at a dose of 10,000 EID. 50 / feather, the incidence and mortality of chickens in each group were observed and counted every day after the infection, and throat swabs were collected to determine the toxin excretion situation.

[0030] 1. Morbidity, mortality, and protection index after challenge The experimental results showed that all chickens in each group were normal after immunization, exhibiting no abnormalities. Post-challenge observations revealed that chickens in the FPV group gradually developed clinical symptoms such as dyspnea, head shaking, and sneezing. Some birds also exhibited depression and anorexia, ultimately leading to death. By the end of the trial, the mortality rate was 8 / 10. In the commercial ILTV vaccine group and the FPV-ILTV-immunized group, the vast majority of birds showed no visible clinical symptoms after challenge, similar to those in the healthy control group. However, two birds in the commercial ILTV vaccine group died, and six birds in the FPV-gB group died (see Table 3). Post-challenge autopsies of surviving and deceased birds revealed normal visceral tissues, including the larynx, trachea, and spleen, in the blank control group. Autopsies of deceased birds in the FPV and FPV-gB-immunized groups revealed congestion and swelling of the larynx and trachea, with significant blood clots in the trachea. The proportion of visceral lesions in the FPV group was 10 / 10; after the chickens immunized with FPV-ILTV and commercial ILTV vaccine were challenged, no obvious pathological changes were found in the parenchymal organs of all the chickens.

[0031] 2. Vaccine protection index = (number of deaths in the challenge control group - number of deaths in the immunized control group) / number of deaths in the challenge control group × 100.

[0032] The results are shown in Table 3. There was no mortality in the FPV-ILTV group, with a protection index of 100; the mortality rate in the FPV-gB group was 60%, with a protection index of 25; the mortality rate in the commercial ILTV vaccine was 20%, with a protection index of 75; and the mortality rate in the FPV control group was 80%. These results indicate that FPV-ILTV can provide strong protection against the attack of virulent ILTV strains and is an ideal candidate for an ILTV vaccine.

[0033] Table 3 Statistics of morbidity and mortality of chickens in each group after challenge

[0034] 3. Detoxification test Throat swabs were collected on days 3, 5, and 7 after challenge, stored in 1 ml of PBS containing double-antibody, and frozen and thawed three times. The supernatant was centrifuged and inoculated into the chorioallantoic membrane of 10-day-old SPF chicken embryos at a rate of 0.2 ml / embryo. After incubation at 37°C for 120 hours, the chorioallantoic membrane was observed for pathological signs such as pox, edema, and delayed embryonic development. This confirmed positive results for ILTV isolation and determined virus shedding. Virus isolation rate = number of chickens with positive laryngeal virus isolation / total number of chickens tested.

[0035] The results, as shown in Table 4, showed that on day 3 post-challenge, no birds in any group died. The virus isolation rate in the FPV-ILTV group was 60%, the virus shedding rate in the commercial ILTV vaccine group was 80%, and the virus shedding rates in the FPV and FPV-gB groups were 100%. On day 5, no birds in the FPV-ILTV group died, while birds in the commercial ILTV vaccine group, the FPV group, and the FPV-gB group died. Furthermore, the virus isolation rates in the FPV-ILTV, commercial ILTV vaccine, and FPV groups were all 100%. Therefore, compared with the FPV and commercial ILTV vaccine groups, the FPV-ILTV group shed less virus and had a lower mortality rate, making it an ideal ILTV vaccine candidate.

[0036] Table 4 Statistical results of virus isolation rate of chickens in each group after challenge

[0037] Example 3. Study on the ratio and immune effect of the dual recombinant fowlpox virus vector vaccine The construction of FPV-AEV recombinant fowlpox virus and the preparation method of vaccine refer to invention patent CN116478939B.

[0038] To study the ratio and immune effect of combined vaccines, the present invention randomly divided 70 one-day-old SPF chickens into seven groups. Groups 1 and 2 were subcutaneously vaccinated with 10,000 PFU / bird of the recombinant two-in-one vaccine FPV-AEV:FPV-ILTV (1:1) at day 1; Group 3 was subcutaneously vaccinated with 10,000 PFU / bird of FPV-AEV at day 1; Group 4 was subcutaneously vaccinated with 10,000 PFU / bird of FPV-ILTV at day 1; and Groups 5 and 6 were subcutaneously vaccinated with 10,000 PFU / bird of FPV at day 1. Twenty-one days after immunization, chickens in Groups 1, 3, and 5 were challenged with a virulent AEV strain via intracerebral inoculation at a dose of 10,000 EID. 50 Chickens in groups 2, 4, and 6 were challenged with a virulent strain of ILTV via the laryngeal inoculation route at a dose of 10,000 EID 50 The chickens were given 1 dose of avian encephalomyelitis virus (AEPV) per bird. Morbidity and mortality in each group were observed and recorded daily after challenge. Blood was collected weekly from chickens in Groups 1, 3, 5, and 7 after immunization to measure antibody levels following immunization using a commercial avian encephalomyelitis virus antibody detection kit. Throat swabs were collected from chickens in Groups 2, 4, 6, and 7 every 1, 3, 5, and 7 days after immunization to determine virus excretion. Group 7 served as a blank control group.

[0039] 1. Morbidity and mortality outcomes The test results (Table 5) show that all groups of chickens were normal after immunization, with no abnormalities. Post-challenge observation revealed that the vast majority of chickens in Groups 1-4 showed no clinical symptoms, while those in Groups 5-6 showed no clinical symptoms. However, chickens in Group 5 gradually developed clinical symptoms such as ataxia, unsteady standing, and depression. Group 6 gradually developed clinical symptoms such as dyspnea, conjunctivitis, head shaking, sneezing, and some chickens also showed depression. Group 7 served as the blank control group, which was not inoculated with the virulent strain and did not exhibit any clinical symptoms.

[0040] Table 5 Statistical results of morbidity and mortality of chickens in each group after challenge

[0041] 2. Antibody change results Blood was collected from chickens in groups 1, 3, 5, and 7 every week after immunization, and the antibody changes after immunization were detected using a commercial avian encephalomyelitis virus antibody detection kit. Figure 5 As shown in Figure 2, no AEV antibodies were detected in the blank control group (Group 7) during the entire immunization period using an AEV antibody detection kit. No AEV antibodies were detected in the FPV control group (Group 5) during the immunization period, but slightly detected in AEV ELISA antibodies 1 week after challenge. In Group 1 FPV-AEV / FPV-ILTV (1:1) and Group 3 FPV-AEV, AEV ELISA antibodies were detected 1 week after immunization, and the antibody levels gradually increased. 3 weeks after challenge, the antibody levels further increased (see Figure 2). Figure 5 ).

[0042] 3. Virus isolation rate results Throat swabs were collected on days 3, 5, and 7 after challenge, stored in 1 ml of PBS containing double-antibody, and frozen and thawed three times. The supernatant was centrifuged and inoculated into the chorioallantoic membrane of 10-day-old SPF chicken embryos at a rate of 0.2 ml / embryo. After incubation at 37°C for 120 hours, the chorioallantoic membrane was observed for pathological signs such as pox, edema, and delayed embryonic development. This confirmed positive results for ILTV isolation and determined virus shedding. Virus isolation rate = number of chickens with positive laryngeal virus isolation / total number of chickens tested.

[0043] The results, as shown in Table 6, showed that on day 3 post-challenge, no birds in any group died. The virus isolation rate in the FPV-ILTV group was 60%, the virus shedding rate in the commercial ILTV vaccine group was 70%, and the virus shedding rate in the FPV group was 100%. On day 5, no birds in the FPV-ILTV group died, while birds in the commercial ILTV vaccine group and the FPV group died. The virus isolation rates in the FPV-ILTV, commercial ILTV vaccine, and FPV groups were all 100%. Therefore, compared with the FPV and commercial ILTV vaccine groups, the FPV-ILTV group shed less virus and had a lower mortality rate, making it an ideal ILTV vaccine candidate.

[0044] Table 6 Statistical results of virus isolation rate of chickens in each group after challenge

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant fowlpox virus, characterized in that The recombinant fowlpox virus is obtained by cloning the P1 and 3C genes of avian encephalomyelitis virus AEV and the UL32 and gB genes of avian infectious laryngotracheitis virus ILTV into a fowlpox virus FPV vector to package the recombinant fowlpox viruses FPV-AEV and FPV-ILTV.

2. A recombinant fowlpox virus live vector vaccine according to claim 1, characterized in that: The vaccine is prepared by mixing the recombinant fowlpox virus FPV-AEV and FPV-ILTV in a certain proportion.

3. The recombinant fowlpox virus according to claim 1, characterized in that The method for constructing the recombinant fowlpox virus FPV-ILTV comprises the following steps: S1. cDNA fragments of the UL32 and gB genes of avian infectious laryngotracheitis virus (ILTV) were obtained by PCR amplification; S2. The amplified UL32 and gB gene fragments were fused to obtain ILTV-UL32-gB; S3. The fowlpox virus early and late promoters were connected to ILTV UL32-gB to obtain an ILTV UL32-gB expression cassette; S4. The ILTV UL32-gB expression cassette was inserted into the pBlue-FPV-244 vector containing the eGFP expression cassette by in-fusion technology to generate the recombinant plasmid pB-FPV-ILTV; S5. The pB-FPV-ILTV plasmid was transfected into CEF cells. After screening for positive clones, the selection gene was deleted using the Cre enzyme to obtain the recombinant fowlpox virus FPV-ILTV. The recombinant fowlpox virus according to claim 3, characterized in that the avian infectious laryngotracheitis virus UL32 and gB genes in step S1 are reversely linked via a promoter.

4. The recombinant fowlpox virus according to claim 3, characterized in that The fowlpox virus early and late promoter in step S3 is PE / L.

5. The recombinant fowlpox virus according to claim 3, characterized in that The fowlpox virus early promoter in step S3 is P7.

5.

6. The recombinant fowlpox virus according to claim 3, characterized in that The nucleotide sequence of the ILTV UL32-gB expression cassette in step S3 is shown in SEQ ID NO.

1.

7. The recombinant fowlpox virus according to claim 3, characterized in that The eGFP expression cassette described in step S4 contains LOXP sequences on both sides.

8. Use of the recombinant fowlpox virus according to claim 1 in the preparation of avian encephalomyelitis virus and avian infectious laryngotracheitis virus vaccines.

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