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Recombinant S1 protein of novel mutant strain of porcine epidemic diarrhea virus and subunit vaccine of recombinant S1 protein

A technology of porcine epidemic diarrhea and mutated strains, applied in antiviral agents, recombinant DNA technology, cells modified by introducing foreign genetic material, etc., can solve the problem of not achieving good immune effect

Active Publication Date: 2014-09-17
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, researchers have tried a variety of methods to study PEDV vaccines, such as DNA vaccines, genetically modified plant vaccines, and recombinant adenovirus vaccines, but none of them have achieved good immune effects

Method used

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  • Recombinant S1 protein of novel mutant strain of porcine epidemic diarrhea virus and subunit vaccine of recombinant S1 protein
  • Recombinant S1 protein of novel mutant strain of porcine epidemic diarrhea virus and subunit vaccine of recombinant S1 protein
  • Recombinant S1 protein of novel mutant strain of porcine epidemic diarrhea virus and subunit vaccine of recombinant S1 protein

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Embodiment Construction

[0046] 1. Materials and methods

[0047] 1.1 Virus strains, plasmids and cells

[0048] E.coil DH5α, Vero cells, PEDV attenuated strain DR13 are preserved by our laboratory. Pseudomonas was purchased from China Veterinary Drug Control Institute. pFastBac in Insect Baculovirus Expression System TM Dual plasmid, pVAX-S1(m) plasmid, Sf9 insect cells and DH10Bac strain were purchased from Invitrogen. The above test materials are conventional materials, which can be purchased from the market by those skilled in the art.

[0049] 1.2 Reagents and experimental animals

[0050] Rabbit anti-S1 protein polyclonal antibody and mouse anti-Pseudomonas exotoxin A protein polyclonal antibody were prepared and preserved in our laboratory (can be prepared by conventional experimental methods). DNA gel recovery kit and Plasmid Miniprep Kit were purchased from BIOMEGA Company. HRP-labeled goat anti-mouse IgG, goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG and goat anti-rabbit Ig...

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Abstract

The invention discloses a recombinant S1 protein of a novel mutant strain of a porcine epidemic diarrhea virus and a subunit vaccine of the recombinant S1 protein. The protein is obtained by the steps of cloning a fusion gene fragment PE(delta III)-S1(m)-KDEL3 containing a KDEL3 gene sequence, a gene sequence of III-region-deleted pseudomonas aeruginosa exotoxin A and an S1(m) gene sequence to a baculovirus vector to obtain a recombinant vector; carrying out recombinant vector site specificity transposition on a seroconversion DH10Bac competent cell of the recombinant vector to obtain a recombinant bacmid; transfecting the recombinant bacmid to an Sf9 cell under the mediation of a liposome to obtain a recombinant Sf9 cell; expressing by using the recombinant Sf9 cell. According to the invention, a PE(delta III)-S1(m)-KDEL3 fusion protein is firstly expressed by using a baculovirus expression system on the basis of optimizing the predilection of a codon of mammalian with S1 genes, so that the recombinant baculovirus for efficiently and accurately expressing the fusion protein is obtained. Proved by IFA and Western blot, the recombinant baculovirus can be used for accurately expressing the fusion protein, and the fusion protein has favorable reactionogenicity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant S1 protein of a new mutant strain of porcine epidemic diarrhea virus and a subunit vaccine thereof. Background technique [0002] Porcine epidemic diarrhea virus (PEDV) is a single-stranded positive-sense RNA virus with an envelope, belonging to type I coronavirus. Like other coronaviruses, the PEDV S protein plays an important role in inducing neutralizing antibodies, binding cells, and promoting virus and cell fusion [2] . Studies have shown that most of the neutralizing epitope regions of the S protein are located in the S1 region; in addition, comparing the PEDV mutant strains and traditional strains, the S protein has the largest variation, and the main variation occurs in the S1 region. [0003] Pseudomonas exotoxin A (PEA) is a cytotoxic exotoxin secreted by Pseudomonas aeruginosa. PEA is composed of 613 amino acids and can be divided into three di...

Claims

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Application Information

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IPC IPC(8): C12N15/50C12N15/63C12N5/10A61K39/215A61P31/14
Inventor 姜平赵攀登白娟
Owner NANJING AGRICULTURAL UNIVERSITY
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