Single-factor serum antigen of IBVS1 polypeptide and application thereof in preparation of recombinant adenovirus

Abstract

The application belongs to the technical field of polypeptide application, and particularly relates to a single-factor serum antigen of IBVS1 polypeptide and application thereof in preparation of recombinant adenovirus, wherein the amino acid sequence of the polypeptide antigen is SEQ ID NO:1 or SEQ ID NO:3. In the application, the S1 subunit closely related to virus serotype specificity and neutralizing antibody induction is selected as an immunogen, so that the problem of non-key antigen interference with immune response in whole virus or multi-antigen vaccine is effectively avoided, and the targeting and effectiveness of vaccine immunization are improved. In addition, the IBV S1 antigen is expressed by using a recombinant adenovirus carrier, so that the recombinant adenovirus carrier not only has the advantages of high expression efficiency, strong immunogenicity and good safety, but also can induce strong humoral immunity and cellular immunity response of the body, thereby enhancing the immune protection effect on IBV infection.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical preparation technology, specifically relating to a single-factor serum antigen of IBVS1 polypeptide and its application in the preparation of recombinant adenovirus. Background Technology

[0002] Infectious bronchitis (IB) is an acute, highly contagious viral disease of chickens caused by infectious bronchitis virus (IBV). IBV was first reported in the United States in 1931; it is a single-stranded positive-sense RNA virus with a genome of 27.6 kb. IBV infection in chickens causes an acute, highly contagious respiratory disease, resulting in significant economic losses to the poultry industry.

[0003] In my country, vaccination is the primary method for preventing IBV infection. However, due to the large number of IBV serotypes, poor cross-protection between serotypes, and the continuous emergence of IBV variants, existing commercial vaccines cannot provide complete immune protection. Therefore, the development of new and highly effective vaccines is of great significance for the prevention and control of IBV. Summary of the Invention

[0004] This invention provides a single-factor serum antigen of IBVS1 polypeptide and its application in the preparation of recombinant adenovirus, thereby overcoming the shortcomings of the prior art.

[0005] This invention first provides a polypeptide antigen S1 of chicken infectious bronchitis virus, the amino acid sequence of which is as follows:

[0006]

[0007] The polypeptide antigen S1 has the following nucleotide sequence:

[0008]

[0009] Furthermore, to improve the expression of the antigen factor in adenovirus, its amino acid sequence was optimized. The optimized amino acid sequence of the antigen factor is as follows:

[0010] (SEQ ID NO:3).

[0011] Its corresponding nucleotide sequence is as follows:

[0012]

[0013] The present invention also provides a recombinant adenovirus, which is prepared by inserting the nucleotide fragment of the above-mentioned antigen factor into an adenovirus.

[0014] The present invention also provides a vaccine prepared using the above-mentioned recombinant adenovirus as an antigen.

[0015] This invention provides an IBV S1 single-factor serum polypeptide antigen and its application in the preparation of a recombinant adenovirus vaccine. By selecting the S1 subunit, which is closely related to viral serotype specificity and neutralizing antibody induction, as the immunogen, the problem of non-critical antigens interfering with the immune response in whole-virus or multi-antigen vaccines is effectively avoided, thus improving the specificity and effectiveness of vaccine immunization. Furthermore, this invention uses a recombinant adenovirus vector to express the IBV S1 antigen, which not only has advantages such as high expression efficiency, strong immunogenicity, and good safety, but also can induce a strong humoral and cellular immune response, thereby enhancing the immune protection against IBV infection. Attached Figure Description

[0016] Figure 1 Antigenicity analysis diagram;

[0017] Figure 2 : Image of PCAGGS-MSS1-His recombinant plasmid double enzyme digestion identification, where M represents Trans 5K DNA Marker; samples 1-4 represent recombinant eukaryotic expression vector PCAGGS-MSS1-His double enzyme digestion samples;

[0018] Figure 3 : Western blotting detection of S1 protein expression, where M represents protein marker; 1, 2 μg transfected with PCAGGS-MSS1-His plasmid; 2, 4 μg transfected with PCAGGS-MSS1-His plasmid; 3, blank control well;

[0019] Figure 4 Single-factor serum identification diagram;

[0020] Figure 5 : Recombinant shuttle plasmid PDC316-MSS1 double enzyme digestion identification, where M: Trans 5K DNA Marker; 1-4: PDC316-MSS1 recombinant plasmid double enzyme digestion samples;

[0021] Figure 6Packaging of recombinant adenovirus in HEK293 cells: A: 3 days after co-transfection of HEK293 cells with recombinant shuttle plasmid and backbone plasmid; B: 3 days after co-transfection of HEK293 cells with recombinant shuttle plasmid and backbone plasmid; C: 3 days after co-transfection of HEK293 cells with recombinant shuttle plasmid and backbone plasmid.

[0022] Figure 7 Observation of cytopathic effects in HEK293 cells infected with recombinant adenovirus, including: A: HEK293 cells infected with recombinant adenovirus rAdv-MSS1; B: HEK293 cells infected with recombinant adenovirus rAdv-GFP; C: negative control.

[0023] Figure 8 :Identification of rAdv-MSS1 target protein expression:

[0024] Figure 9 Growth curves of recombinant adenovirus rAdv-MSS1 and rAdv-GFP;

[0025] Figure 10 Figure 1 shows the amplification results of the target gene of recombinant adenovirus rAdv-MSS1 at different generations; where M represents the Trans 2K DNA Marker; 1 represents the amplification results of the target gene of recombinant adenovirus rAdv-MSS1 at generation P2; 2 represents the amplification results of the target gene of recombinant adenovirus rAdv-MSS1 at generation P4; 3 represents the amplification results of the target gene of recombinant adenovirus rAdv-MSS1 at generation P6; 4 represents the amplification results of the target gene of recombinant adenovirus rAdv-MSS1 at generation P8; and 5 represents the amplification results of the target gene of recombinant adenovirus rAdv-MSS1 at generation P10.

[0026] Figure 11 : rAdv-MSS1 recombinant adenovirus antibody titer chart;

[0027] Figure 12 : Protection rate of rAdv-MSS1 recombinant adenovirus as measured. Detailed Implementation

[0028] The SPF chicken embryos and SPF chickens used in this embodiment were purchased from Jinan Spafars Poultry Co., Ltd.; MSS1 single-factor serum was preserved in our laboratory; adenovirus shuttle plasmid PDC316-EGFP and adenovirus backbone plasmid pBHGlox(delta)E1, 3Cre were purchased from Zhuangmeng Biotechnology. The S1 gene sequence of the IBV mutant strain ahysx-1 was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and ligated into the PUC57 vector, named IBV-MSS1-PUC57.

[0029] The adenovirus vector used in this invention is one of the most commonly used vaccine vectors, characterized by high expression levels and the ability to induce specific humoral and cellular immune responses. It also boasts advantages such as broad host range and ease of manipulation, making it widely used in recombinant vaccine research. This invention uses adenovirus as a vector to insert the isolated IBV S1 single-factor serum antigen nucleic acid fragment into the adenovirus vector, successfully constructing recombinant adenovirus rAdv-MSS1, laying the foundation for subsequent vaccine research on chicken infectious bronchial virus (IBV).

[0030] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0031] Example 1: Screening and Optimizing Antigen Factors and Preparing S1 Gene Single-Factor Serum

[0032] The S gene (amino acid sequence SEQ ID NO:1, corresponding nucleotide sequence SEQ ID NO:2) was analyzed, and the S1 gene, which has high antigenicity, was extracted based on its antigenicity. This extracted portion can facilitate the expression of recombinant proteins and improve their expression characteristics. Figure 1 Figure A shows a schematic diagram of antigen protein sequence optimization, and Figure B shows an antigenicity analysis diagram of the antigen protein. Through antigenicity analysis of the full-length sequence and the truncated S1 sequence, it can be seen that the antigenicity of the truncated S1 (amino acid sequence is SEQ ID NO:3, and its corresponding nucleotide sequence is SEQ ID NO:24) is significantly improved, and IBV hysx-1 S1 gene single-factor serum was prepared.

[0033] 1) Inoculate the IBV-MSS1-PUC57 bacterial culture into 5 mL of LB solution containing ampicillin antibiotic, place it in a constant temperature shaking incubator at 220 r / min, 37℃ overnight, and extract the IBV-MSS1-PUC57 plasmid.

[0034] 2) The target fragment MSS1 was amplified by PCR using a high-fidelity enzyme. Xho I and Bgl II restriction enzyme sites were added to the upstream and downstream primers, respectively, and a 6×His tag sequence was introduced downstream of S1. After the reaction, the amplification product and negative control were detected by 1% agarose gel electrophoresis. After confirmation, the PCR product and pCAGGS vector were double-digested and ligated using T4 enzyme to construct the recombinant plasmid. Positive colonies were selected for identification, and the recombinant plasmid was extracted and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. After successful sequencing, the recombinant plasmid was preserved for subsequent experiments.

[0035] The S1 gene was identified by double digestion using the designed restriction endonucleases Xho I and Bgl II in the recombinant single-factor serum plasmid PCAGGS-MSS1-His. The double digestion yielded the expected target fragment and a vector fragment of approximately 4800 bp. Figure 2 ).

[0036] 3) The in vitro expression characteristics of the successfully constructed PCAGGS-MSS1-His recombinant plasmid were identified. When the 293T cells reached a confluence density of 95%, the cells were digested and seeded into six-well plates, which were then incubated overnight at 37°C in a 5% CO2 incubator. When the cell density in the six-well plates reached 80%, the recombinant plasmid was transfected into 293T cells, and after 72 hours of culture, cell samples were collected to identify the expression of MSS1 protein.

[0037] Transfected 293T cells were collected, and lysed proteins were analyzed by Western blotting to detect the expression of S1 protein in 293T cells. Results showed that the lysed protein from cells transfected with the recombinant plasmid PCAGGS-MSS1-His exhibited a specific band at 100 kDa, consistent with the expected molecular weight of the S1 protein; both this sample and the blank control showed specific bands at 27 kDa, consistent with the expected molecular weight of the internal control GAPDH protein. Figure 3 ).

[0038] 4) When the PCAGGS-MSS1-His recombinant plasmid was identified in vitro as being able to effectively express the S1 protein, the plasmid was extracted by removing endotoxin and injected into 21-day-old SPF chickens via intramuscular injection in the leg (100 μg / chick). A booster immunization was given 14 days after the first immunization, and blood was collected from the heart 21 days after the first immunization. The serum was then separated and stored at -20°C.

[0039] The IBV variant ahysx-1 virus was cultured and concentrated to obtain a high-titer viral load. The virus was then denatured to prepare Western blotting samples. After Western blotting, collected single-factor serum was used as the primary antibody for identification. Western blotting results showed a specific band at 100 kDa, comparable to the expected molecular weight of the S1 protein. This serum specifically bound to the IBV variant ahysx-1 S1 protein, but not to other IBV strains, such as GI-19. This indicates that the prepared single-factor serum specifically binds to the ahysx-1 S1 protein without binding to the S1 protein of other genotypes, and can be used for subsequent identification of IBV ahysx-1 virus. Figure 4 ).

[0040] Example 2: Construction and Identification of rAdv-MSS1

[0041] 1. Construction of PDC316-MSS1 recombinant plasmid

[0042] The target fragment MSS1 was amplified by PCR using a high-fidelity enzyme. EcoR I and Bgl II restriction enzyme sites were introduced at the 5' and 3' ends of the upstream and downstream primers, respectively. After the reaction, the amplification product and negative control were detected by 1% agarose gel electrophoresis. After confirmation, the PCR product and PDC316-EGFP vector were double-digested and ligated using T4 enzyme to construct the recombinant plasmid. Positive colonies were selected for identification, and the recombinant plasmid was extracted and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. After successful sequencing, the recombinant plasmid was preserved for subsequent experiments.

[0043] The S1 gene of IBVahysx-1 was identified by double digestion of the adenovirus recombinant shuttle plasmid PDC316-MSS1 with restriction endonucleases EcoR I and Bgl II. The target fragment and the vector fragment of PDC316 were obtained after double digestion. Figure 5 ).

[0044] 2. Rescue and identification of rAdv-MSS1 recombinant virus

[0045] 1) Plasmid preparation: The rescue of recombinant adenovirus consists of a two-plasmid system: PDC316-MSS1-EGFP vector is an adenovirus shuttle vector of the Ad Max system and pBHGlox(delta)E1,3Cre vector is an adenovirus backbone vector of the Ad Max system (which can undergo homologous recombination under the action of Cre / lox P recombinase in 293 cells for adenovirus packaging). The two plasmids were detoxified and extracted separately. After the concentration was measured, they were stored at -20℃ for later use.

[0046] 2) Rescue of recombinant adenovirus: One day before transfection, 293 cells were digested and seeded into six-well plates at an appropriate ratio. When the cell density reached 80-90%, the cells were transfected with plasmids. The shuttle plasmid PDC316-MSS1-EGFP and the adenovirus backbone plasmid pBHGlox(delta)E1,3Cre were prepared. Take two 1.5 mL sterile centrifuge tubes and add 200 μL of opti-MEM culture medium to each. Add 9 μL of lip3000 to one tube and 2 μg of shuttle plasmid, 4 μg of viral backbone plasmid, and 12 μL of lip3000 to the other. Combine the mixtures from both centrifuge tubes into one tube, gently mix, and incubate at room temperature for 20 min. Discard the culture medium in the six-well plate and wash the cells three times with PBS. Add the above mixture to the two wells and incubate the six-well plate at 37°C with 5% CO2 for 12 h. Replace with DMEM containing 2% FBS. Culture for 5-7 days; observe and record cell growth daily. When the cells exhibit typical pathological changes (enlarged, rounded cells, and cells clustering in ordinary chains), store the six-well plate at -80°C and freeze-thaw three times to collect the viral fluid.

[0047] HEK293 cells were co-transfected with an adenovirus backbone and shuttle plasmid using a transfection reagent and cultured for 5-7 days. Cell status was observed every 24 hours. When most cells detached from the six-well plate and showed typical cytopathic effects, the packaged product was harvested and passaged for recombinant virus culture. Figure 6 ).

[0048] When packaged primary (P0 generation) recombinant adenovirus was used to infect normal HEK293 cells, cytopathic effects such as cell swelling, rounding, grape-like appearance, and detachment from the cell culture dish were observed. Control group cells, however, did not exhibit these changes. Figure 7 ).

[0049] 3) Western blot detection of target protein expression: The collected recombinant adenovirus was inoculated into 293 cells at a ratio of 1:100 and cultured for 72-96 h. Cell samples were then collected and protein samples were prepared. The primary antibody was the single-factor serum prepared for this study, which was diluted with 2% BSA solution at a ratio of 1:20 and incubated overnight. Horseradish peroxidase (HRP)-labeled goat anti-chicken IgG secondary antibody was diluted 1:10000 with 2% skim milk and mixed thoroughly. The PVDF membrane, which had been washed with PBST, was placed in the secondary antibody solution and incubated on a shaker for 2 h. After incubation with the secondary antibody, the PVDF membrane was rinsed with TBST solution for 30 min, with the TBST being replaced every 10 min. The rinsed PVDF membrane was then blotted dry with absorbent paper and placed in a petri dish. An equal volume of chromogenic solution was gently mixed in, and the mixture was allowed to stand at room temperature for 1 min. The petri dish containing the PVDF membrane was then placed in the imaging system, and the prepared luminescent mixture was dropped onto the membrane surface. The program was opened, the exposure time was set to 1 s, and the image was taken and saved.

[0050] Forty-eight hours after infecting cells with the recombinant adenovirus rAdv-MSS1, infected cell samples were collected and prepared into protein samples. Western blotting was used to detect the expression of the IBV ahysx-1 mutant S1 protein in HEK293 cells. The results showed that the recombinant adenovirus rAdv-MSS1 exhibited a specific band at approximately 100 kDa, while rAdv-GFP showed no band, consistent with previous experimental results. Figure 8 ).

[0051] HEK293 cells were infected with purified and concentrated recombinant adenovirus using serial dilutions. After 48 hours of infection, the percentage of positive wells was calculated based on cytopathic effects, and the titer was determined. The results showed that the titer of rAdv-MSS1 was 1.878 × 10⁻⁶. 10 PFU / mL; the titer of rAdv-GFP was 5.634 × 10⁻⁶. 10 PFU / mL.

[0052] Dilute the recombinant adenovirus to 1×10⁻⁶. 5 HEK293 cells were infected with PFU / mL. Virus was harvested at 24 h, 36 h, 48 h, 60 h, and 72 h post-infection, and its titer was determined. The measured viral titers were plotted as curves. The results showed that the growth curve of recombinant adenovirus rAdv-MSS1 was consistent with that of the control adenovirus rAdv-GFP, with no significant difference. Figure 9 ).

[0053] 4) Genetic stability of rAdv-MSS1

[0054] The packaged primary recombinant adenovirus was used to infect normal HEK293 cells. When most cells showed CPE, the cells were collected, and the cells were repeatedly frozen and thawed three times before being passaged again. The recombinant adenoviruses from generation P1 to generation P10 were harvested sequentially. The nucleic acids of the adenoviruses from generation P2, P4, P6, P8, and P10 were extracted, and the loss of the target gene was detected using the target gene primer PDC316-F / R.

[0055] After infecting 293 cells with rAdv-MSS1 recombinant adenovirus, the cells were passaged for 10 consecutive generations. Adenovirus nucleic acid was extracted from generations P2, P4, P6, P8, and P10. The target gene was amplified using primers, and the sequence was sequenced. The results showed the appearance of a target band consistent with the expected fragment size. Figure 10 ).

[0056] 5) Evaluation of rAdv-MSS1 immunogenicity

[0057] One experimental group and one challenge control group were set up. The experimental group was immunized with rAdv-MSS1 at 10 6 TCID 50 SPF chickens were immunized with a titer of 1 mL / mL via intranasal administration, while the control group was immunized with only 1 mL of DMEM. Blood samples were collected at 7, 14, and 21 days post-immunization, and serum was separated. Intranasal challenge was administered 21 days post-immunization with a challenge dose of 10 mL / mL. 5 EID 50 / Only.

[0058] The identified rAdv-MSS1 recombinant adenovirus was administered via intranasal immunization to 14-day-old SPF chickens. Antibody titers induced in the chickens were consistently higher than 10 on days 7-28 post-immunization. 2.5 The virus was challenged 21 days after the initial immunization, resulting in a protection rate of 50%, while the mortality rate in the challenged control group was 100%. Figure 11 , 12 ).

[0059] In summary, the S1 single-factor antigen described in this invention has a clear molecular composition and stable immune properties, which is beneficial for vaccine quality control and large-scale production. It can overcome, to some extent, the prevention and control challenges caused by the diversity of IBV serotypes, poor cross-protection, and frequent occurrence of variant strains, providing a novel, efficient, and promising technical solution for the immunization and prevention of IBV.

Claims

1. A single-factor serum antigen of an IBV S1 polypeptide, characterized in that, The amino acid sequence of the serum antigen is SEQ ID NO:

3.

2. The serum antigen as described in claim 1, characterized in that, The nucleotide sequence of the serum antigen is SEQ ID NO:

4.

3. The use of the serum antigen according to claim 1 in the preparation of recombinant adenovirus.

4. A recombinant adenovirus, characterized in that, The recombinant adenovirus is prepared by inserting a nucleotide fragment encoding the serum antigen of claim 1 into an adenovirus.

5. A vaccine, characterized in that, The vaccine comprises the recombinant adenovirus as described in claim 4.

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