Recombination and expression for non-antibiotic expression vector of rotavirus Vp6 gene and lactic acid bacteria

A rotavirus and expression vector technology, applied in viral peptides, genetic engineering, plant genetic improvement, etc., can solve problems such as environmental microecological damage

Inactive Publication Date: 2007-04-25
JILIN AGRICULTURAL UNIV +3
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  • Abstract
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  • Claims
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Problems solved by technology

However, due to the continuous drift and diffusion of drug resistance genes into the environment, it may cause damage to the environmental microecology and bring serious consequences. Therefore, this type of expression system is still far from the requirement of "edibility" and cannot be directly used in humans. and animals

Method used

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  • Recombination and expression for non-antibiotic expression vector of rotavirus Vp6 gene and lactic acid bacteria

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Embodiment 1

[0080] 1. PCR amplification and cloning of the target gene

[0081] According to the gene sequence of (AF317123.) porcine rotavirus Wa strain Vp6 in GenBank, design a pair of PCR primers (synthesized by Dalian TaKaRa company) that amplifies this gene: P 1 : 5'-GTG GAGCTC ATGGAG GTT CTG TAC TCT TTA T-3';P 2 : 5'-GTG GGTACC TCA CTT AAT CAACAT GCT TCT-3' (the underlined sequences are SacI and KpnI restriction sites respectively). The total RNA of porcine rotavirus was extracted according to the method of Trizol kit; the Vp6 gene was amplified by RT-PCR using the total RNA as template and P1 and P2 as primers. The 50 μl reverse transcription reaction system and reaction conditions are as follows: take 10 μl mRNA, heat at 75°C for 5 minutes to denature the double-stranded RNA, and quickly place it on ice to cool, then add 10.0 μl of 5×AMV Buffer; 4.0 μl of dNTPs (10 mM each); RNasin 1.0μl; downstream primer 0.5μl; AMV (5Units / μl) 0.5μl; Olig(dT) 1.0μl; MgCl 2 (25mM) 4.0μl; ...

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Abstract

The invention belongs to the field of biological gene, disclosing the recombination and expression of a rotavirus Vp6 gene and Bacterium acidi lactic nonresistance expression vector and it is characterized in: digesting the cloning vector and vector taking thyA gene as a selection pressure using Sac I and KpnI, purifying and recovering, linking the products with T4 DNA ligase, transforming to thyA gene-deficient E. coli competence E. coli X13, culturing in the medium, and screening to obtain prokaryotic recombinant expression plasmid through growing functional redeem, inducing positive bacteria for expression using threonine to obtain high expression of the recombinant plasmid. Molecular weight of fusion protein is about 44.88 KD. Beneficial effects are: VP6 protein is RV-group-specific antigen, locating at the virus endoconch, occupying 51%of the virus particles. As VP6 mainly stimulates organism to generate mucosal immune antibody sIgA, it plays a very important role in the mucosal immune, thus advanced development of rotavirus Vp6 mediated gene oral mucosal immune genetically engineered vaccine has important significance.

Description

technical field [0001] The invention belongs to the field of biological genes, and in particular relates to the gene sequence of the Vp6 gene of the Wa strain of porcine group A rotavirus, the cloning of the Vp6 gene, and "vaccine-grade" non-antibiotic-resistant ones that can shuttle and express between lactic acid bacteria and Escherichia coli Plasmid vector pW425t recombination, Lactobacillus, mucosal immunity, expression in Escherichia coli. Background technique [0002] Rotavirus (RV) is one of the main pathogens causing non-bacterial diarrhea in infants and various young animals. RV diarrhea is a worldwide infectious disease. Since Mebus et al first isolated rotavirus (RV) from calf diarrhea in 1969, people have gradually found that the virus is the main pathogen that causes diarrhea and death in infants and young animals. Infections caused by rotavirus kill nearly one million infants and young children every year in developing countries. Group A RV is the most impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/70C12N15/46C07K14/14
Inventor 王春凤王会岩李玉
Owner JILIN AGRICULTURAL UNIV
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