Recombinant lactobacillus casei for expressing infectivity pancreatic necrosis virus VP2 protein and production method thereof

A technology of Lactobacillus casei and necrosis virus, which is applied in the field of recombinant Lactobacillus casei and its production method, and the preparation of biological products, which can solve the problems of no biological activity, inability to transfer antigenic components, and pyrogens caused by toxic cells

Inactive Publication Date: 2009-08-19
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

Escherichia coli is the most widely used host bacteria for expressing heterologous proteins, because Escherichia coli grows fast and has good representative genetic characteristics, it is still widely used to express heterologous proteins, but in Escherichia coli The expressed fusion protein exists in the form of insoluble inclusion bodies and has no biological activity, and Escherichia coli contains toxic cellular pyrogens, so it

Method used

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  • Recombinant lactobacillus casei for expressing infectivity pancreatic necrosis virus VP2 protein and production method thereof
  • Recombinant lactobacillus casei for expressing infectivity pancreatic necrosis virus VP2 protein and production method thereof
  • Recombinant lactobacillus casei for expressing infectivity pancreatic necrosis virus VP2 protein and production method thereof

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Embodiment Construction

[0036] The present invention is described in more detail below in conjunction with accompanying drawing example:

[0037] 1. Construction of IPNV VP2 gene Lactobacillus casei expression vector

[0038] The recombinant plasmid pMD18-T-IPNV VP2 and plasmid pPG1 were extracted from Escherichia coli pMD18-T-IPNV VP2 / JM109 and Lactobacillus casei393 containing plasmid pPG1 with a plasmid DNA mini-extraction kit. After pPG1 and pMD18-T-IPNV VP2 were double digested, the target fragments were recovered and purified. The recovered purified products of pPG1 and VP2 were ligated, and the ligated products were electrotransformed into competent cells Lactobacillus casei 393, and the Lactobacillus casei containing the positive recombinant plasmid pPG1-IPNV VP2 was named pPG1-IPNV VP2 / L.casei 393.

[0039] 2. Induced expression of IPNV VP2 protein in Lactobacillus casei

[0040] Inoculate a single colony of pPG1-IPNV VP2 / L.casei 393 into 5mL MRS liquid medium for overnight culture to acti...

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Abstract

The invention provides a recombinant Lactobacillus casei for expressing the infectious pancreas necrosis virus (IPNV) VP2 protein and a preparation method thereof. According to the whole gene sequence of the IPNV VP2 protein and the gene fusion characteristic of expression vector plasmid, a pair of primers are designed to perform PCR to obtain target segment of 1095bp containing the main antigen site of the IPNV VP2 protein, the IPNV VP2 protein obtained through amplification is linked to the surface expressed carrier plasmid pPG1 and enters the cell of the host strain Lactobacillus casei 393 through electrotransformation, and the Lactobacillus casei containing positive recombinant plasmid pPG1-IPNV VP2 is named pPG1-IPNV VP2/L. casei 393. The recombinant pPG1-IPNV VP2/L. casei 393 is expressed under the induction of lactose. In the invention, the Lactobacillus casei expression carrier system of the IPNV VP2 protein is constructed and the target protein of around 40 KDa containing the main antigen site of the IPNV VP2 protein is expressed, and according to the Western blot experiment and indirect immunofluorescence experiment, the expressed foreign protein is capable of reacting with the IPVN immune serum, which shows that the recombinant VP2 protein and the IPNV natural antigen have the same antigenicity.

Description

(1) Technical field [0001] The present invention relates to a biological product, and the present invention also relates to a preparation method of the biological product. Specifically, it is a recombinant Lactobacillus casei expressing infectious pancreatic necrosis virus VP2 protein and a preparation method. (2) Technical background [0002] Infectious pancreatic necrosis is a highly contagious and acute viral disease of salmonid fish caused by infectious pancreatic necrosis virus (IPNV). The typical symptoms are abnormal swimming and dark and dark body color of sick fish , abdominal distention, exophthalmos, hemorrhage and necrosis of subcutaneous, hepatopancreas and other visceral parenchymal organs. Eggs excrete pathogens and become a potential source of infection. Due to the widespread prevalence of the disease and the high morbidity and mortality rates, it has brought significant economic losses to the salmon and trout breeding industry in my country and the world. ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/34C12N15/74C12R1/245
Inventor 刘敏李一经葛俊伟乔薪瑗赵丽丽
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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