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Swine transmissible gastroenteritis S/N protein fusion gene, recombinant lactococcus lactis and application

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 carcinogenesis, and achieve ideal mucosal immune protection and strengthen the immune system. Immunogenic effects

Inactive Publication Date: 2012-04-04
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 S/N protein fusion gene, recombinant lactococcus lactis and application
  • Swine transmissible gastroenteritis S/N protein fusion gene, recombinant lactococcus lactis and application
  • Swine transmissible gastroenteritis S/N protein fusion gene, recombinant lactococcus lactis and application

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Effect test

Embodiment 1

[0028] Construction and identification of embodiment 1 MLSN gene Lactococcus lactis secretion expression vector

[0029] 1 Design and synthesis of multi-antigen epitope sequence MLSN

[0030] Porcine transmissible gastroenteritis virus S protein contains four antigenic sites A, B, C, and D, of which 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 in vivo, that is, specific antibodies against S protein.

[0031] Through two flexible Linkers ((GGGGS) 3 ) the A, D antigen site genes and N of TGEV 321 The antigenic site genes are connected (the sequence is D-Linker-N 321 -L...

Embodiment 2

[0071] Expression and identification results of embodiment 2 MLSN gene Lactococcus lactis

[0072] Construction and electrotransformation of Lactococcus lactis with 1MLSN gene

[0073] Mix the ligation product with electroporation 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, The electric shock parameters are voltage 2kV, time 4.5ms; add 900μL ice-precooled SGM17MC (GM17 liquid medium, add 0.5mol / L sucrose, 0.02mol / L magnesium chloride, 0.002mol / L calcium chloride) after electric shock to restore the culture transfer the bacterial solution to a 1.5mL centrifuge tube, and place it on ice for 10 minutes; incubate anaerobically at 30°C for 2 hours; take an appropriate amount of bacterial solution and spread it on GM17 agar medium containing 5 μg / mL chloramphenicol, 30 ℃ anaerobic culture for 2-3d. Pick a single colon...

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Abstract

The invention discloses a swine transmissible gastroenteritis S / N protein fusion gene, recombinant lactococcus lactis and application. The invention provides an antigen fusion gene obtained by connecting A and D antigen loci in swine transmissible gastroenteritis virus (TGEV) S protein and an N321 antigen locus in N protein and a streptococcus pyogenes cell wall anchoring sequence, and a nucleotide sequence of the antigen fusion gene is shown as SEQ ID No. 1. The invention further provides an expression vector containing the antigen fusion gene and the recombinant lactococcus lactis strain containing the recombinant expression vector. The recombinant lactococcus lactis strain is induced by using nisin, the fusion gene can be expressed on the surface of the bacterial cell wall. Immunoblotting experiments indicate that the expressed recombinant protein can react with TGEV immune serum and has the same antigenicity with TGEV natural antigens, and the recombinant protein can be prepared into safe and effective mucous immune live vaccines 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 bacterial strains and their application 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...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/63C12N1/21A61K48/00A61K39/225A61P31/14C12R1/01
Inventor 张金林马立保胡晓芬
Owner WUHAN HUAYANG ANIMAL PHARMA
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