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Porcine epidemic diarrhea S1 protein fusion gene, recombinant bacillus megaterium strain and application

A technology of porcine epidemic diarrhea and gene fusion, applied in gene therapy, recombinant DNA technology, bacteria, etc., can solve the problems of ineffective induction of slgA, strong virus virulence, and resistance to infection, so as to maintain the balance of intestinal micro-ecosystem , reduce colonization, and stabilize the plasmid

Inactive Publication Date: 2012-04-04
WUHAN HUAYANG ANIMAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Conventional inactivated vaccines and attenuated vaccines have played an active role in the prevention and treatment of these two diseases and achieved good results, but there are still some problems, such as poor immunogenicity of inactivated vaccines, which cannot effectively induce slgA and resist infection; attenuated vaccines After the animal body sheds the virus, the virulence of the virus may become stronger, etc. In addition, it must be immunized by injection

Method used

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  • Porcine epidemic diarrhea S1 protein fusion gene, recombinant bacillus megaterium strain and application
  • Porcine epidemic diarrhea S1 protein fusion gene, recombinant bacillus megaterium strain and application
  • Porcine epidemic diarrhea S1 protein fusion gene, recombinant bacillus megaterium strain and application

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Effect test

Embodiment 1

[0029] Example 1MLS 1 Construction and identification of secretory expression vector of Bacillus megaterium

[0030] 1 epitope sequence MLS 1 design and synthesis of

[0031] According to the complete PEDV gene sequence AF353511 registered in Genbank, find out the 22-505aa sequence encoding the S glycoprotein, and use the flexible Linker ((GGGGS) 3 ) to the S of PEDV 1 Antigenic site gene and cell wall anchor sequence (CWA M6 ) are connected, and the sequence is named MLS 1 (SEQ ID No. 1). Changes to rare codons such as threonine (ACG), histidine (CAC), arginine (CGG, CGA), alanine (GCG), and leucine (CTC, CTG), but not their encoded amino acid. in MLS 1 BglII restriction site and SphI restriction site were introduced upstream and downstream respectively. The designed MLS 1 The sequence was artificially synthesized, and the recombinant plasmid pMD18-MLS was constructed 1 .

[0032] pMD18-T Simple Vector was purchased from TaKaRa (Dalian) Company, pMD18-MLS 1 The s...

Embodiment 2

[0071] Example 2MLS 1 Transformation and expression identification of gene Bacillus megaterium

[0072] 1 Preparation of Bacillus megaterium protoplasts

[0073] Pick a ring of Bacillus megaterium (Bacillus megaterium) WH320 (purchased from MoBiTec Company) to streak on the LB plate, culture overnight at 37°C, pick a single colony from the above plate and inoculate it into 1×AB3 liquid medium, 200r / Cultivate overnight at 37°C; take 1ml of the above-mentioned seed liquid and transfer it to a 250ml Erlenmeyer flask filled with 50ml 1×AB3 medium, cultivate at 200r / min at 37°C until OD600≈1.0, collect the bacteria by centrifugation, add 5ml of SMMP solution to suspend the bacteria; Transfer the solution into a 100ml Erlenmeyer flask, add lysozyme with a final concentration of 0.1mg / ml, place it in a water bath at 37°C for 30 minutes, and observe the formation of protoplasts with a microscope, so that the formation of protoplasts is about half; centrifuge for 10 minutes to collect ...

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Abstract

The invention discloses a porcine epidemic diarrhea S1 protein fusion gene, recombinant bacillus megaterium strain and application. An antigen fusion gene shown as SEQ ID No. 1. is obtained by connecting an antigen locus of porcine epidemic diarrhea virus (PEDV) S glycoprotein and a cell wall anchoring sequence. The invention further constructs a secretion expression vector containing the antigen fusion gene, and secretion expression vector is converted into the bacillus megaterium to express recombinant protein on the cell wall or surface of the bacillus megaterium. Immunoblotting experiments indicate that the expressed recombinant protein can react with PEDV immune serum and has the same antigenicity as PEDV natural antigens. Immunofluorescent tests of live bacteria which are subjected to induced expression indicate that the expressed recombinant protein is positioned to the surfaces of the bacteria. Experiment results indicate that the recombinant protein can be prepared into safe and effective mucous immune live vaccines for preventing and treating porcine epidemic diarrhea.

Description

technical field [0001] The invention relates to an antigen fusion gene and a recombinant strain containing the antigen fusion gene, in particular to porcine epidemic diarrhea S 1 A protein fusion gene and an expression vector containing the fusion gene. The present invention further relates to recombinant Bacillus megaterium strains containing the expression vector and their application in preparing vaccines for preventing and treating porcine epidemic diarrhea, belonging to the field of porcine epidemic diarrhea prevention and control. Background technique [0002] Porcine epidemic diarrhea (PEDV) is an acute highly contagious infectious disease of pigs caused by coronavirus, characterized by watery diarrhea, vomiting and dehydration. to major economic losses. The virus mainly infects pigs through the intestinal tract, and has obvious characteristics of intestinal tissue tropism. In addition to humoral immunity and cellular immunity, the local intestinal mucosal immune sys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/63C12N1/21A61K48/00A61K39/215A61P31/14C12R1/11
Inventor 张金林马立保胡晓芬
Owner WUHAN HUAYANG ANIMAL PHARMA
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