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Porcine epizootic diarrhea virus S1 protein fusion gene and recombinant bacillus megaterium, and their use

A porcine epidemic diarrhea, fusion gene technology, applied in gene therapy, recombinant DNA technology, bacteria and other directions, can solve the problem of ineffective induction of slgA, virus virulence, resistance to infection, etc., to maintain the balance of the intestinal micro-ecosystem , the effect of reducing colonization and plasmid stabilization

Active Publication Date: 2013-07-10
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 epizootic diarrhea virus S1 protein fusion gene and recombinant bacillus megaterium, and their use
  • Porcine epizootic diarrhea virus S1 protein fusion gene and recombinant bacillus megaterium, and their use
  • Porcine epizootic diarrhea virus S1 protein fusion gene and recombinant bacillus megaterium, and their use

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 MLS 1 Construction and identification of secretory expression vector of Bacillus megaterium

[0034] 1 epitope sequence MLS 1 design and synthesis of

[0035] 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 genes and cell wall anchor sequences (CWA M6 ) concatenated, 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 .

[0036] pMD18-T Simple Vector was purchased from TaKaRa (Dalian) Company, pMD18-MLS 1 The speci...

Embodiment 2

[0077] Example 2MLS 1 Transformation and expression identification of gene Bacillus megaterium

[0078] 1 Preparation of Bacillus megaterium protoplasts

[0079] Pick a ring of Bacillus megaterium (Bacillus megaterium) WH320 (purchased from MoBiTec Company) and streak it 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 seed solution 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 P...

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Abstract

The invention discloses a porcine epizootic diarrhea virus S1 protein fusion gene and recombinant bacillus megaterium, and their use. An S1 antigen site of an S glycoprotein of the porcine epizootic diarrhea virus is connected to an anchor sequence of a cell wall so that the porcine epizootic diarrhea virus S1 protein fusion gene shown in the formula of SEQ ID No.1 is obtained. The invention further discloses a secrete expression vector containing the porcine epizootic diarrhea virus S1 protein fusion gene. The secrete expression vector containing the porcine epizootic diarrhea virus S1 protein fusion gene is transferred into the bacillus megaterium and a recombinant protein is expressed on the cell wall or the surface of the bacillus megaterium. A result of a western blot experiment shows that the expressed recombinant protein can react with PEDV immune serum so that it is proved that the expressed recombinant protein has the antigenicity the same as antigenicity of the PEDV natural antigen. A post-inducible expression living bacterium immuno-fluorescence test proves that the expressed recombinant protein is positioned on the surface of the recombinant bacillus megaterium. An experiment result shows that the recombinant protein provided by the invention can be prepared into a safe and effective mucosal immune live bacterium vaccine for preventing and treating porcine epizootic 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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