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New genetic engineering subunit vaccine for transmissible gastroenteritis virus of swine

An infectious and gastroenteritis technology, applied in genetic engineering, viruses, viral peptides, etc., can solve the problems of lack of glycosylation modification of antigenic proteins, strong virulence of virus strains, and weak immune protection, etc., to achieve protein immunity Good originality, batch-to-batch stability, and good immunogenicity

Inactive Publication Date: 2019-07-23
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

However, inactivated vaccines have weak autoimmune protection and incomplete inactivation, resulting in the risk of spreading the virus; attenuated live vaccines have the risk of the virulence of the strain becoming stronger
[0005] The patent CN 103194471A application discloses a double-gene lactic acid bacteria vaccine containing a segment of the porcine transmissible gastroenteritis virus S1 gene and an N gene, but the immunogenicity is poor
Patent CN 104740626B discloses a vaccine for preventing porcine transmissible gastroenteritis. This patent uses recombinant Salmonella as a carrier. Salmonella is prokaryotic expression. The antigenic protein lacks the necessary glycosylation modification, and the immunogenicity is poor. Immunization to Salmonella has a large side effect , and there is a risk of strong return of Salmonella

Method used

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  • New genetic engineering subunit vaccine for transmissible gastroenteritis virus of swine
  • New genetic engineering subunit vaccine for transmissible gastroenteritis virus of swine
  • New genetic engineering subunit vaccine for transmissible gastroenteritis virus of swine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1 Construction of recombinant eukaryotic expression vector pCI-S1-GS

[0103] 1. The codon-optimized S1 gene was obtained from Nanjing GenScript Biotechnology Co., Ltd., and 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.

[0104] 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.3 and 4), 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.

[0105] Table 1 S1 gene amplification system

[0106]

[0107]

[0108] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such as figure 1 As...

Embodiment 2

[0119] Example 2: Construction and screening of recombinant CHO cells

[0120] 1. Cell Transfection

[0121] 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.

[0122] 1.2 Plasmid and cell mixing Take 5ug of the pCI-S1-GS plasmid vector in Example 1, add it to the EP tube, add 0.7ml cells, mix well, and let stand for 15 minutes.

[0123] 1.3 Electric transfer to 280V 20ms for 2 pulses. After the electric shock is completed, immediately transfer the cells to a shaker flask for suspension culture. After 48 hours,...

Embodiment 3

[0133] Example 3 SDS-PAGE detection

[0134] The cell culture supernatant harvested in Example 2 was subjected to SDS-PAGE detection, and empty CHO cells were used as a negative control. The specific operation is as follows: take 40 μl of harvested cell culture, add 10 μl of 5×loading buffer, bathe in boiling water for 5 minutes, centrifuge at 12000 r / min for 1 minute, take the supernatant and carry out SDS-PAGE gel (12% concentration gel) electrophoresis, After electrophoresis, the gel was stained and decolorized to observe the target band. Such as Figure 4 As shown, the target band appears around the molecular weight of about 150kDa. Because the S1 protein has more glycosylation modifications, its molecular weight is about 66kDa higher than the molecular weight calculated by amino acid sequence theory. The negative control has no band at the corresponding position.

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Abstract

The present application provides an immunological composition and a subunit vaccine. The immunological composition comprises: a swine transmissible gastroenteritis virus S1 protein encoded by a nucleic acid molecule of SEQ ID NO:1 or a nucleic acid molecule of 95% or more identical to the nucleotide sequence of the SEQ ID NO:1. The vaccine uses an eukaryotic expression of CHO cells, is sufficientin protein glycosylation, good in antigen protein immunogenicity, and also very high in expression levels, and reaches 2-3 g / L. The recombinant cells can be subjected to suspension cultivation in a large scale, which greatly reduces complexity of vaccine preparation and saves production costs.

Description

technical field [0001] The application relates to the technical field of animal immunity drugs, in particular to a novel genetically engineered subunit vaccine of porcine transmissible gastroenteritis virus. Background technique [0002] Porcine transmissible gastroenteritis (Transmissible Gastroenteritis, TGE) is a highly contagious digestive tract infectious disease of pigs caused by porcine transmissible gastroenteritis virus. The disease is characterized by vomiting, watery diarrhea and dehydration, and it can lead to death in severe cases. Pigs of different breeds and ages are susceptible to the disease, especially piglets within 2 weeks of age can have a mortality rate as high as 100%. OIE lists it as a Category B animal disease. [0003] Porcine transmissible gastroenteritis virus of Swine (TGEV) is a member of the genus Coronaviridae of the family Coronaviridae. There are three main structural proteins of TGEV: spike glycoprotein (S or E2), membrane structural prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/50A61K39/225A61P31/14
CPCA61K39/225A61P31/14C07K14/005C12N2770/20022C12N2770/20034
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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