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Porcine epidemic diarrhea virus genetic engineering vaccine and preparation method thereof

A technology of genetic engineering vaccines and porcine epidemic diarrhea, applied in genetic engineering, antiviral immunoglobulin, botanical equipment and methods, etc., can solve the problems of limited capacity, affecting particle formation, affecting secondary structure, etc.

Active Publication Date: 2019-03-12
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is worth noting that the human hepatitis B core antigen has a limited capacity for inserting antigenic sequences as a protein carrier, and the insertion of larger-length antigenic sequences into the protein carrier will affect its secondary structure, thereby affecting the formation of particles (Borisova, G.1996, Intervirology, Vol.39, p.16-22)

Method used

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  • Porcine epidemic diarrhea virus genetic engineering vaccine and preparation method thereof
  • Porcine epidemic diarrhea virus genetic engineering vaccine and preparation method thereof
  • Porcine epidemic diarrhea virus genetic engineering vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Construction of fusion gene fragment HBcAg-PEDV-COE

[0029] 1. Construction of fusion gene fragment HBcAg-PEDV-COE: as figure 1 As shown, using molecular biology techniques and methods, the PEDV COE gene is inserted into the gene encoding the HBcAg (human hepatitis B core antigen) molecule, and the inserted position is the nucleotide sequence encoding the 79th amino acid and encoding the 80th amino acid In between, the fusion gene fragment HBcAg-PEDV-COE (SEQ ID NO.8) was constructed.

[0030] 2. Ligation of the target gene with the transfer plasmid: Digest the insect expression vector pFastBac I and HBcAg-PEDV-COE gene amplified fragments with BamH I and Hind I respectively, recover and purify them, and ligate them overnight at 4°C with T4 DNA ligase.

[0031] 3. Transformation of the ligation product into competent cells: transform the ligation product into T1 competent cells under sterile conditions, specifically, mix well, ice-bath for 30 minutes, he...

Embodiment 2

[0037] Embodiment 2: Preparation of recombinant HBcAg-PEDV-COE protein

[0038]1. Recombinant baculovirus amplification: Inoculate the recombinant baculovirus rBac-HBcAg-PEDV-COE into insect cells sf9, culture at 27°C for 4 days, collect the culture, centrifuge and take the supernatant to obtain the f2 generation recombinant baculovirus;

[0039] 2. Identification of expressed protein:

[0040] (1) Inoculate the insect cell sf9 with the inoculum amount of the f2 generation recombinant baculovirus at MOI=5-10, culture at 27°C for 4 days, collect the culture, centrifuge and take the supernatant to obtain the recombinant VP3 protein;

[0041] (2) SDS-PAGE identification: The above supernatant was subjected to SDS-PAGE electrophoresis; after electrophoresis, after staining and decolorization, it was found that the molecular weight was consistent with the theoretical size at about 32kDa, indicating that the expression was successful.

[0042] (2) Western Blot identification: Take ...

Embodiment 3

[0046] Example 3 Preparation of Porcine Epidemic Diarrhea Virus Genetic Engineering Subunit Vaccine

[0047] Take the purified HBcAg-PEDV-COE recombinant protein obtained in Example 2, add an adjuvant to emulsify, mix well, and store at 4°C. See Table 1 for specific vaccine ratios.

[0048] Table 1 Porcine epidemic diarrhea virus genetically engineered subunit vaccine composition ratio

[0049]

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Abstract

The invention discloses a porcine epidemic diarrhea virus genetic engineering vaccine and a preparation method thereof, which belongs to the field of veterinary biological products. Core proteins of hepatitis B virus are used as a vector, a porcine epidemic diarrhea virus protective antigen is inserted between a 79 amino acid to 80 amino acid of the vector by using a molecular biology method to form the genetic engineering vaccine for preventing the porcine epidemic diarrhea virus. The preparation method of the vaccine is simple, a great amount of antigen proteins can be prepared, theconsumed time is short, the expression amount is high, the production cost is greatly reduced, and the mass production is facilitated. The porcine epidemic diarrhea sub-unit vaccine comprising HBcAg-PEDV-COE recombinant proteins prepared in the invention is good in immunity effect, small in immunity dose, and capable of effectively preventing the infection of the porcine epidemic diarrhea virus.

Description

technical field [0001] The invention relates to a porcine epidemic diarrhea virus genetic engineering vaccine and a preparation method thereof, belonging to the field of veterinary biological products. Background technique [0002] Porcine epidemic diarrhea (PED) is an intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), and its main clinical features are watery diarrhea, vomiting and dehydration. The disease was first discovered in Europe in the 1970s, and then became widespread worldwide, causing huge economic losses to the pig industry. The disease mainly causes fatal diarrhea in newborn piglets, and the fatality rate even reaches 100%. [0003] The S glycoprotein of PEDV carries the main B lymphocyte antigen epitope, which is the main structural protein that induces the body to produce neutralizing antibodies and provides immune protection. Experiments have confirmed that the PEDV S protein contains multiple antigenic epitope sequences. Using...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/866C12N15/62A61K39/215A61P31/14C07K16/10G01N33/569
Inventor 丁国伟范娟钱钟叶正琴潘杰李玉和宋庆庆
Owner 扬州优邦生物药品有限公司
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