Swine transmissible gastroenteritis virus S/N protein fusion gene and recombinant lactococcus lactis, and their use

A fusion gene and infectious technology, applied in gene therapy, recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of strong virulence, uncontrolled expression, and somatic cell canceration, and achieve ideal mucosal immune protection and strengthen the immune system. Immunogenic effects

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

The use of live carriers such as bacteria and viruses to deliver intact antigenic components solves this problem. At present, there are reports of using attenuated bacteria as carriers of vaccine antigens, such as Salmonella, attenuated Listeria, etc., but Salmonella may exist as a carrier to deliver DNA vaccines The risk of strong virulence, plasmid DNA may also be integrated into the host cell genome, causing a series of problems such as gene disorder that regulates cell growth, uncontrolled expression of proto-oncogenes and tumor suppressor genes, somatic cell canceration, and cell transformation.

Method used

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  • Swine transmissible gastroenteritis virus S/N protein fusion gene and recombinant lactococcus lactis, and their use
  • Swine transmissible gastroenteritis virus S/N protein fusion gene and recombinant lactococcus lactis, and their use
  • Swine transmissible gastroenteritis virus S/N protein fusion gene and recombinant lactococcus lactis, and their use

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Experimental program
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Embodiment 1

[0031] Construction and identification of embodiment 1 MLSN gene Lactococcus lactis secretion expression vector

[0032] 1 Design and synthesis of multi-antigen epitope sequence MLSN

[0033] The S protein of porcine transmissible gastroenteritis virus contains four antigenic sites, A, B, C, and D, among which the A (536-593 residue region) and D (376-392 residue region) antigenic sites are induced Neutralizing antibodies play an important role, and the A antigenic site is a major B cell epitope. Porcine transmissible gastroenteritis virus contains 4 main T cell epitopes, of which the N on the N protein 321 (321-335 residue region) antigenic site can induce the strongest T cell response, and can induce T cells to assist in the synthesis of neutralizing antibodies, that is, specific antibodies against S protein, in vivo.

[0034] Through two flexible Linkers ((GGGGS) 3 ) the A, D antigen site genes and N of TGEV 321 The antigenic site genes are connected (the order is D-Lin...

Embodiment 2

[0074] Expression and identification results of embodiment 2 MLSN gene Lactococcus lactis

[0075] Construction and electrotransformation of Lactococcus lactis with 1MLSN gene

[0076] Mix the ligation product with electrotransformation competent cells NZ9000 (Lactococcus lactis NZ9000 purchased from MoBiTec) and place on ice for 5 min; transfer it to a 2 mm pre-cooled electroporation cup; electroporate with Transformation Apparatus 165-2101, Electric shock parameters are voltage 2kV, time 4.5ms; after electric shock, add 900 μL ice-precooled SGM17MC (GM17 liquid medium, add 0.5mol / L sucrose, 0.02mol / L magnesium chloride, 0.002mol / L calcium chloride) to resume culture and mix well; transfer the bacterial solution to a 1.5mL centrifuge tube, place on ice for 10min; incubate anaerobically at 30°C for 2h; take an appropriate amount of bacterial solution and smear it on GM17 agar medium containing 5μg / mL chloramphenicol, 30 ℃ anaerobic culture for 2-3d. Pick a single colony on t...

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Abstract

The invention discloses a swine transmissible gastroenteritis virus S / N protein fusion gene and recombinant lactococcus lactis, and their use. A and D antigen sites of a S protein of the swine transmissible gastroenteritis virus and an N321 antigen site of an N protein of the swine transmissible gastroenteritis virus are connected to a cell wall anchor sequence of streptococcus pyogenes so that the swine transmissible gastroenteritis virus S / N protein fusion gene is obtained and has a nucleotide sequence shown in the formula SEQ ID No.1. The invention further discloses an expression vector containing the swine transmissible gastroenteritis virus S / N protein fusion gene and the recombinant lactococcus lactis containing the expression vector. The recombinant lactococcus lactis is induced by Nisin so that the swine transmissible gastroenteritis virus S / N protein fusion gene can be expressed on the bacterial cell wall surface. A result of a western blot experiment shows that the expressed recombinant protein can react with TGEV immune serum so that it is proved that the expressed recombinant protein has the antigenicity the same as antigenicity of the TGEV natural antigen and thus the expressed recombinant protein can be prepared into a safe and effective mucosal immune live bacterium vaccine for preventing and treating swine transmissible gastroenteritis.

Description

technical field [0001] The present invention relates to an antigen fusion gene and a recombinant strain containing the antigen fusion gene, in particular to a porcine transmissible gastroenteritis virus S / N protein fusion gene and an expression vector containing the fusion gene, and the present invention further relates to a recombinant strain containing the expression vector The invention relates to Lactococcus lactis strains and their use in preparing vaccines for preventing and treating porcine transmissible gastroenteritis, belonging to the field of prevention and treatment of porcine transmissible gastroenteritis. Background technique [0002] Porcine transmissible gastroenteritis is a highly contagious disease caused by transmissible gastroenteritis virus of swine (TGEV), characterized by severe diarrhea, vomiting, dehydration and high mortality. TGEV mainly exists in the jejunum and duodenum, and pigs of various ages are susceptible. Because piglets have relatively p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/63C12N1/21A61K48/00A61K39/225A61P31/14C12R1/46
Inventor 张金林马立保胡晓芬
Owner WUHAN HUAYANG ANIMAL PHARMA
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