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Genetic engineering subunit vaccine of avian infectious bronchitis

A chicken infectious and bronchitis technology, applied in genetic engineering, vaccines, veterinary vaccines, etc., can solve the problems of low antigen expression, easy to cause pollution, difficult chicken embryos, etc., achieve high expression, low production cost, The effect of easy quality control

Pending Publication Date: 2019-07-09
苏州世诺生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing patents are almost all whole virus inactivated vaccines or live vaccines. For example, the IBV vaccine disclosed in CN103007272A is a live vaccine. The whole virus is produced by suspension cell culture, which requires serum and has the risk of spreading the virus, and the antigen expression is low; at the same time Existing vaccines are produced from chicken embryos, which are difficult to handle and easily cause pollution
CN104353070A discloses a genetically engineered subunit vaccine of chicken infectious bronchitis virus and its preparation method, which uses the fusion expression of chicken bronchitis virus H120 strain S1 gene and H3N2 influenza virus HA2 gene, and uses insect baculovirus expression system to carry out preparation, but there is a big difference in the glycosylation of insect cells and animal cells

Method used

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  • Genetic engineering subunit vaccine of avian infectious bronchitis
  • Genetic engineering subunit vaccine of avian infectious bronchitis
  • Genetic engineering subunit vaccine of avian infectious bronchitis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Embodiment 1 Construction of recombinant eukaryotic expression vector pCI-S1-GS

[0114] 1. The codon-optimized S1 gene was obtained from Nanjing GenScript Biotechnology Co., Ltd., and the S1 gene was cloned into the pUC-57 vector to construct the pUC-S1 plasmid vector. The optimized S1 gene sequence is shown in SEQ ID NO.1.

[0115] 2. S1 gene amplification uses pUC-S1 as a template, and S1-F and S1-R as primers for PCR amplification (the gene sequences of S1-F and S1-R are shown in SEQ ID NO.6 and 7), and the amplified See Table 1 for the augmentation system. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0116] Table 1 S1 gene amplification system

[0117]

[0118]

[0119] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such...

Embodiment 2

[0130] Example 2 Construction of recombinant eukaryotic expression vector pCI-S1-S2-GS.

[0131] 1. The codon-optimized S2 gene expression cassette (sequence shown in SEQ ID NO: 5) is from Nanjing GenScript Biotechnology Co., Ltd. (the expression cassette contains a CMV promoter, the truncated S2 gene (sequence is shown in SEQ ID NO: 5) : 2) and the SV40 polyA transcription termination signal, and cloned into the pUC-57 vector to construct the pUC-S2 plasmid vector.

[0132] 2. Amplification of the S2 gene expression cassette Use pUC-S2 as a template, and S2-F and S2-R as primers (the gene sequences of S2-F and S2-R are shown in SEQ ID NO.8 and 9) for PCR amplification , see Table 5 for the amplification system. The reaction conditions are: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 4 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0133] Table 5 S2 gene e...

Embodiment 3

[0147] Example 3: Construction and screening of recombinant CHO-S cells

[0148] 1. Cell Transfection

[0149] 1.1 Prepare cells Take CHO cells in the logarithmic growth phase, sample and count, and use 1×10 6 The cell density of cells / ml continues to be subcultured, maintain the seeds, centrifuge the remaining cells, centrifuge at 1000rpm for 4 minutes, discard the supernatant, resuspend with about 20ml of fresh CHO-WM medium, centrifuge again, centrifuge at 1000rpm for 4 minutes, discard the supernatant After resuspending with a small amount of medium for counting, the final cell density was adjusted to 1.43×10 7 cells / ml.

[0150] 1.2 Plasmid and cell mixing Take 5ug of the pCI-S1-S2-GS plasmid vector in Example 2, add it to the EP tube, add 0.7ml cells, mix well, and let stand for 15 minutes.

[0151] 1.3 Electrotransformation 280V20ms electric shock for 2 pulses, after the electric shock is completed, immediately transfer the cells to a shaker flask for suspension cult...

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Abstract

The invention provides a genetic engineering subunit vaccine of the avian infectious bronchitis. The genetic engineering subunit vaccine of the avian infectious bronchitis comprises avian infectious bronchitis virus S1 protein and S2 protein coded respectively by nucleic acid molecules shown as SEQ ID NO:1 or nucleic acid molecules identical with more than 95% of a nucleic acid sequence of SEQ IDNO:1 and nucleic acid molecules shown as SEQ ID NO:2 or nucleic acid molecules identical with more than 95% of a nucleic acid sequence of SEQ ID NO:2. A heterodimer structure can be formed between theS1 protein and the S2 protein and is similar to a virus surface structure, the S1 protein and the S2 protein are in eukaryotic expression and are sufficient in protein glycosylation, high in antigenprotein immunogenicity and quite high in expression quantity reaching 2-3g / L, recombinant cells are supportive of large-scale suspension culture, vaccine preparation complexity is lowered greatly, andproduction cost is lowered.

Description

technical field [0001] The application relates to the technical field of animal immunization drugs, in particular to a genetically engineered subunit vaccine for chicken infectious bronchitis. Background technique [0002] Infectious bronchitis (Avian Infectious Bronchitis, IB) is an acute, highly contagious respiratory and urogenital disease of chickens caused by Avian Infectious Bronchitis Virus (IBV). Rales, coughing, and sneezing are the main features. Chickens of various ages, genders and breeds are susceptible, but chicks under 6 weeks of age are more susceptible. Infection of infectious bronchitis virus in chicks that have not acquired maternal antibodies can cause permanent oviduct damage, loss of egg-laying ability at sexual maturity, and even death due to respiratory or kidney infection. Infectious bronchitis virus infection of laying hens leads to decreased egg production, irregular, deformed and low-quality eggs, and reduced fertilization rate. Infectious bronc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215A61P31/14C07K14/165C12N15/50
CPCA61K39/12A61P31/14C07K14/005A61K2039/552C12N2770/20022C12N2770/20034
Inventor 曹文龙孔迪滕小锘易小萍张大鹤
Owner 苏州世诺生物技术有限公司
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