Recombination Lactococcus lactics for expressing pig infectious gastroenteritis virus S protein and preparation method thereof

A kind of Lactococcus lactis, infectious technology, applied in the field of preparation of biological products, can solve the problem of unsatisfactory immune protection effect

Inactive Publication Date: 2009-12-09
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Practice has proved that the secretory antibody (sIgA) produced by intestinal mucosal immunization is an effective antibody against TGEV infection, while other serum antibodies produced by oral immunization, such as IgG and IgM, are not ideal for the immune protection of this disease

Method used

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  • Recombination Lactococcus lactics for expressing pig infectious gastroenteritis virus S protein and preparation method thereof
  • Recombination Lactococcus lactics for expressing pig infectious gastroenteritis virus S protein and preparation method thereof
  • Recombination Lactococcus lactics for expressing pig infectious gastroenteritis virus S protein and preparation method thereof

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Embodiment Construction

[0022] The present invention is described in more detail below in conjunction with accompanying drawing example:

[0023] 1. Construction of TGEV S gene Lactococcus lactis expression vector

[0024] Using the plasmid pUC-S as a template, using TS7 and TS8 as primers to amplify the fragment containing the main antigenic sites of TGEV S gene A, B, C and D, and named it Sa, the Sa gene was digested with the pNZ8112-Sa vector plasmid, Connected and transformed into competent cell NZ9000 by electrotransformation, the Lactococcus lactis containing the recombinant plasmid was named pNZ8112-Sa / NZ9000.

[0025] 2. Induced expression of TGEV S protein in Lactococcus lactis

[0026] The pNZ8112-Sa / NZ9000 positive recombinant bacteria and the pNZ8112 / NZ9000 empty plasmid control bacteria were inoculated in GM17 liquid medium, and after anaerobic culture at 30°C overnight, the overnight culture was inoculated in 20ml medium at a volume ratio of 1:25. Carry out expansion culture, culture ...

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Abstract

The invention provides a method for expressing the recombinant Lactococcus lactis of porcine transmissible gastroenteristis virus (Transmissible gastroenteristisvirus, TGEV) spike S protein. According to the complete gene sequence of the porcine transmissible gastroenteritis virus S protein and the gene fusion characteristics of the expression vector plasmid, a pair of primers were designed and PCR was carried out to obtain a 2007bp target fragment containing the four main antigenic sites of the TGEV S gene, which was combined with The vector plasmid pNZ8112 for secreted expression was connected, and transformed into host bacteria Lactococcus lactis NZ9000 cells by electrotransformation, and expressed under the induction of nisin. In the present invention, a coronavirus TGEV S protein Lactococcus lactis expression vector system is constructed, and the target protein of about 66KDa containing four main antigenic sites of TGEV S protein A, B, C, and D is expressed, and western blot experiments and indirect immunofluorescence The tests all showed that the expressed foreign protein could react with TGEV immune serum, indicating that the recombinant S protein had the same antigenicity as the TGEV natural antigen.

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. (2) Background technology [0002] Porcine transmissible gastroenteritis (TGE) is a digestive tract infectious disease characterized by piglet vomiting, severe diarrhea and high lethality caused by porcine transmissible gastroenteritis virus (TGEV) of the Coronaviridae family. . The disease is one of the important diseases of early death of piglets in my country and various pig-raising countries in the world. Vaccination is the main measure to prevent this disease. Practice has proved that the secretory antibody (sIgA) produced by intestinal mucosal immunization is an effective antibody against TGEV infection, while other serum antibodies produced by oral immunization, such as IgG and IgM, are not ideal for the immune protection of this disease . Therefore, it is of great significance to choose a carri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/33C12N15/09C07H21/04C12N15/74G01N33/53
Inventor 唐丽杰李一经葛俊伟欧笛
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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