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Recombinant salmonella containing porcine epidemic diarrhea virus S499-789 gene

A Salmonella, recombinant bacteria technology, applied in the field of genetic engineering, can solve the problems of long antigen gene, difficult to guarantee safety, poor strain stability, etc., and achieve the effects of good stability, good protection and good safety.

Inactive Publication Date: 2015-06-03
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, Xu Lili et al. constructed an attenuated Salmonella recombinant vaccine that can express the COE region and SD region of PEDV S protein. The antigen gene contained in it is longer, and the strain stability is worse. At the same time, it contains the crp virulence gene, so the safety is difficult to guarantee.

Method used

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  • Recombinant salmonella containing porcine epidemic diarrhea virus S499-789 gene
  • Recombinant salmonella containing porcine epidemic diarrhea virus S499-789 gene
  • Recombinant salmonella containing porcine epidemic diarrhea virus S499-789 gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Construction of recombinant bacteria of the present invention

[0047] 1. Construction method

[0048] 1PEDV S 499-789 gene cloning

[0049] 1.1 Primer design

[0050] With reference to the S gene cDNA sequence (sequence number: KC886306) of PEDV published by GenBank, primers were designed using Primer5.0 as follows ( Table 1 ), where P1 / P3 are used to amplify S 499-789 Gene. Primers were synthesized by Dalian Bao Biological Engineering Co., Ltd.

[0051] S 499-789 Gene amplification primer sequence and restriction site marking

[0052]

[0053] 1.2 PEDV S 499789 gene amplification

[0054] 1.2.1 Extraction of PEDV RNA

[0055] Use the purified PEDV SC-L strain virus solution frozen in the laboratory as the material, and extract the viral RNA according to the instructions of the total RNA extraction kit. The operation method is as follows:

[0056] (1) Take 250 μL of virus liquid into a 2 mL EP tube, add 750 μL of RZ lysate, and beat several ...

experiment example 1

[0170] Experimental example 1 Characteristic identification of recombinant attenuated Salmonella of the present invention

[0171] 1. Experimental method

[0172] Get the recombinant Salmonella SL7207 (pVAX-S) prepared in Example 1 499-789 ), according to the following method for identification:

[0173] 1 Biological characteristics of recombinant attenuated Salmonella

[0174] 1.1 Determination of growth curve of recombinant attenuated Salmonella

[0175] Pick SL7207 (pVAX-S 499-789 ) single colony, inoculated in 5mL LB liquid medium containing 100μg / mL Kan, until the bacterial solution OD 600 When the value is 0.6, inoculate 1 mL into 100 mL LB liquid medium containing 100 μg / mL Kan, culture at 37 °C, 100 r / min, for 24 h, absorb a small amount of bacterial liquid every 2 h, serially dilute and spread on three On the LB solid plate containing 100 μg / mL Kan, after culturing at 37°C for 18 hours, the colonies were counted, and the growth curves of different strains of reco...

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Abstract

The invention discloses a nucleotide sequence shown in SEQ ID NO:1, and discloses a recombinant carrier and recombinant bacteria containing the nucleotide sequence. A specific antibody generated by immunization of the recombinant salmonella containing the S499-789 gene for the porcine epidemic diarrhea virus is high in level; and the recombinant salmonella has a good protecting effect on a porcine epidemic diarrhea disease and is excellent in clinical application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a 499-789 Genetically recombinant Salmonella. Background technique [0002] Porcine epidemic diarrhea virus is the causative agent of porcine epidemic diarrhea disease (Porcine epidemic diarrhea, PED). There are yellow curds. PEDV is mainly transmitted through the fecal-oral route. After the piglets in the farrowing house ingest the virus through the digestive tract, the virus mainly invades the epithelial cells of the small intestinal mucosa in the jejunum and ileum, and begins to replicate. The infection of PEDV leads to the decrease of the activity of disaccharidase (lactase, sucrase, maltase), aminopeptidase and alkaline phosphatase on the surface of epithelial cell membrane; at the same time, the normal structure of intestinal villi epithelial cells is destroyed and cell fragmentation occurs, resulting in intestinal The atrophy of the villi and the reduction of the inte...

Claims

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Application Information

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IPC IPC(8): C12N15/50C12N15/74C12N15/70C12N1/21C07K14/165C12R1/42C12R1/19
Inventor 黄小波曹三杰陈杰文心田梁恩涛文翼平伍锐张雨迪段素芬朱书权
Owner SICHUAN AGRI UNIV
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