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Genetic engineering subunit vaccine for porcine epidemic diarrhea viruses

A porcine epidemic diarrhea and virus technology, applied in genetic engineering, antiviral agents, plant gene improvement, etc., can solve the problems of strong virulence of virus strains, weak immune protection, incomplete inactivation, etc., and achieve protein immunogen Good sex, good immunogenicity, stable effect between batches

Inactive Publication Date: 2019-06-21
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

Method used

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  • Genetic engineering subunit vaccine for porcine epidemic diarrhea viruses
  • Genetic engineering subunit vaccine for porcine epidemic diarrhea viruses
  • Genetic engineering subunit vaccine for porcine epidemic diarrhea viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Embodiment 1 Construction of recombinant eukaryotic expression vector pCI-S1-GS

[0107] 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.

[0108] 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.

[0109] Table 1 S1 gene amplification system

[0110]

[0111] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such as figure 1 As shown, a ba...

Embodiment 2

[0123] Example 2: Construction and screening of recombinant CHO cells

[0124] 1. Cell Transfection

[0125] 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.

[0126] 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.

[0127] 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

[0137] Example 3 SDS-PAGE detection

[0138] 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×loadingbuffer, 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, electrophoresis Afterwards, the gel was stained and decolorized to observe the target band. The detection result of culture in embodiment 2 is as image 3 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 63kDa 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 invention provides an immunological composition and a genetic engineering subunit vaccine. The immunological composition comprises porcine epidemic diarrhea virus protein S1 encoded by a nucleic acid molecule of SEQ ID NO: 1 or by a nucleic acid molecule 95% or more identical to the SEQ ID NO: 1 in the nucleotide sequence. The vaccine is subjected to eukaryotic expression by CHO (Chinese hamster ovary) cells, the protein glycosylation is sufficient, the immunogenicity of antigen protein is high, and the expression quantity is very high, reaching 2-3g / L; suspension cultivation of recombinant cells can be conducted on a large scale, the complexity of vaccine preparation is greatly reduced, and the production cost is reduced.

Description

technical field [0001] The application relates to the technical field of animal immunization drugs, in particular to a porcine epidemic diarrhea virus genetically engineered subunit vaccine. Background technique [0002] Porcine Epidemic Diarrhea (PED) is an intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV) with vomiting, diarrhea and dehydration as the main symptoms. The disease is susceptible to pigs of all ages, especially piglets within 7 days of age, and the mortality rate after infection is extremely high. [0003] PEDV is a member of the genus Coronavirus in the family Coronaviridae, and only one serotype has been found so far. PEDV is an enveloped, single-stranded positive-sense RNA virus belonging to type I coronaviruses. PEDV S protein is a 20nm spherical rod-shaped glycoprotein protruding from the viral particle envelope, with a molecular weight of about 180-220kDa and composed of about 1383 amino acids. This protein plays an importa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215A61P31/14C07K14/165C12N15/50
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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