Salmonella flagellin fusion protein as well as encoding gene and application thereof

A Salmonella and fusion protein technology, applied in the field of genetic engineering, can solve the problems of mismatched vaccine strains and easy mutation of antigens

Inactive Publication Date: 2017-03-22
INNER MONGOLIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem of the mismatch between the prevailing strains and vaccine strains brought about by the variable characteristics of Salmonella antigens has become increasingly prominent. Therefore, it is urgent to develop a Salmonella "universal vaccine" that can produce cross-immune protection

Method used

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  • Salmonella flagellin fusion protein as well as encoding gene and application thereof
  • Salmonella flagellin fusion protein as well as encoding gene and application thereof
  • Salmonella flagellin fusion protein as well as encoding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0041] The selection and optimization of embodiment 1 gene

[0042] The Salmonella typhimurium flagellin gene (FliC) 1521bp and the cholera enterotoxin B subunit gene (CTB) 375bp were selected, and the codons in the above two genes were transformed into potato-preferred codons through a codon preference library. The analyzed genes were translated into protein sequences using DNA star software, and protein alignment analysis was performed by pBLAST in NCBI. After the alignment is consistent, the splicing and construction of the gene is carried out, and the enhancing and stabilizing sequences are added before and after the fusion sequence, and the required enzyme cutting sites are added at both ends.

Embodiment 2

[0043] The fusion and construction of embodiment 2 gene

[0044] The above two genes were spliced, and the restriction sites were analyzed with Mapdraw. Finally, add the Kozak sequence before the fused FliC and CTB gene sequences, then add the endoplasmic reticulum retention peptide, and add the required enzyme cutting sites at both ends, upstream Ndel, Sal1, and downstream Sac1. The optimized gene was sent to the company for synthesis, and the full length of the synthetic gene was 1932bp. The synthetic sequence was linked into T vector pMD19(Simple), and finally T-Vector pMD19(simple)-fliC-CTB was synthesized. The base sequence of the fusion gene is shown in SEQ ID NO.2, its ORF is 1908bp long, and the base sequence is shown in SEQ ID NO.3. The gene encodes 635 amino acids, including a stop codon.

Embodiment 3

[0045] Embodiment 3 plant expression vector construction

[0046] T-Vector pMD19(simple)-fliC-CTB recombinant plasmid and pRI 101-AN DNA empty vector were respectively digested with Ndel+Sac1 double enzymes, 1% agarose electrophoresis, the voltage was 3-5v / cm, the target fragment FliC- After CTB was separated from the pRI 101-ANDNA vector, it was separated and cut with a surgical blade under ultraviolet light, and the target fragment and vector were recovered with an agarose recovery kit. The target fragment and the carrier were ligated with T4 ligase at an equimolar ratio of 1:1. The ligation reaction was carried out at 16°C for 1 hour, and the reaction was terminated at 70°C. Transform the ligation product into E. coli DH5α, spread 200 μL of the transformed bacteria solution on the flat plate containing kanamycin LB, spread it evenly with a spreader, and culture it horizontally in a 37°C incubator for 1 hour, then turn the plate upside down Incubate for 12 hours. The clon...

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Abstract

The invention relates to the technical field of gene engineering, in particular to salmonella flagellin fusion protein as well as an encoding gene and an application thereof. The salmonella flagellin fusion protein FliC-CTB has the amino acid sequence shown as SEQ ID NO.1. As for the gene fliC-CTB, the salmonella flagellin fusion protein FliC-CTB is encoded and has the base sequence shown as SEQ ID NO.2 or SEQ ID NO.3. By means of a plant genetic transformation technology, a salmonella fliC gene and a cholera enterotoxin B subunit gene are fused and transferred into potatoes, and a transgenic plant is obtained through sieving, integration, transcription and expression of a target gene are proved on the molecular level, the fact that fusion protein has antigen activity through exogenous fusion protein detection of the plant, and the potatoes can be taken as a bioreactor to express the fusion protein by the aid of a fusion gene plant expression vector. A 'general plant oral vaccine' for salmonella can be continuously researched and developed, and a novel effective solution is provided for prevention and treatment of a salmonella disease.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular, the invention relates to a Salmonella flagellar fusion protein, its encoding gene and application. Background technique [0002] The causative agent of salmonellosis belongs to Enterobacteriaceae, Gram-negative enterobacteriaceae. More than 1800 species have been discovered. Salmonella not only causes poultry infection, but also causes bacterial infection in humans, which in turn leads to food poisoning, acute gastroenteritis and other diseases, which are listed as Class B diseases by the International Veterinary Bureau. At present, the European Union, the United States, the United Kingdom, France, Japan, etc. stipulate that Salmonella in meat, eggs, and dairy livestock and poultry products must not be detected. However, as far as the current situation is concerned, most livestock and poultry farms in China use drug control and elimination measures to eliminate Salmon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/82A01H5/00A61K39/112A61K39/39A61P31/04
CPCA61K39/00C07K14/255C07K2319/04C07K2319/55C12N15/8258C12N2800/22Y02A50/30
Inventor 王利张俊霞贾宇臣李少伟
Owner INNER MONGOLIA MEDICAL UNIV
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