Unlock instant, AI-driven research and patent intelligence for your innovation.

Prokaryotic secreation vector for expressing infused E.coli heat-labile enterotoxin B subunit

A fusion expression technology of Escherichia coli, which is applied in the field of prokaryotic expression of Escherichia coli thermosensitive toxin B subunit carrier, can solve the problems such as the difficulty of purification of expression products, and achieve the effect of overcoming the difficulty of expression and purification of expression products and convenient connection

Inactive Publication Date: 2007-08-29
EAST CHINA UNIV OF SCI & TECH
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the prokaryotic expression of LT-B is mostly in the form of inclusion bodies, which brings difficulties to the purification of the expression product (target protein LT-B)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Prokaryotic secreation vector for expressing infused E.coli heat-labile enterotoxin B subunit
  • Prokaryotic secreation vector for expressing infused E.coli heat-labile enterotoxin B subunit
  • Prokaryotic secreation vector for expressing infused E.coli heat-labile enterotoxin B subunit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Construction of pET-LTB:

[0032] The pET32a expression vector constructed by NOVGE is currently a prokaryotic vector widely used in molecular biology and genetic engineering laboratories. The initiation codon ATG, and NdeI (CATATG) containing this codon and the first restriction site Noc I (CCATGG) in the multiple cloning site region are used as the insertion site of the LT-B gene, and These two sites also do not exist in the LT-B gene, which also removes some unnecessary sequences in pET32a, and inserts the LT-B gene sequence containing the signal peptide sequence into pET32a to obtain pET-LTB expression carrier. The specific construction method is as follows:

[0033] 1. Primer design:

[0034] Design and synthesize the following primers according to amplification needs and restriction sites to be introduced:

[0035] Upstream primer P1: 5'GCG CAT ATG AAT AAA GTA AAA TGT TAT GTT-3' (introduce Nde I restriction site);

[0036] Downstream primer P2: 5'GTC CC...

Embodiment 2

[0065] Fusion and secretory expression of target genes EGFP and LT-B

[0066] 1. Insertion of the target gene

[0067] Taking the insertion of the model protein—enhanced green fluorescent protein (EGFP) gene as an example, the EGFP gene constructed on pGEM-T-egfp was digested with EcoR I and XhoI, and the same enzyme was used for the vector pET-LTB Double enzyme digestion, and then the digested gene and vector were connected into a pET-LTB / EGFP expression vector, and then transformation, induced expression and other operations were the same as those described in Example 1.

[0068] 2. PCR identification of expression plasmid

[0069] Taking the EGFP gene as an example, use the purified pET-LTB / EGFP as a template, use Pegfp1 as a forward primer, and Pegfp2 as a reverse primer for PCR amplification. The amplification system is: 10 μl of 10× amplification buffer containing magnesium ions , 4×dNTPs (10mmol) 1μl, upstream and downstream primers (Pegfp1 and Pegfp2) 1μl each, the a...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

This invention relates to a carrier which is applied in the prokaryotic secretion and is expressed combining with the escherichia coli heat-labile enterotoxin B subunit (LT-B), this carrier is a closed loop plasmid,it uses the pET32a as its bed rim, and it is constructed by inserting the nucleic acid list of the escherichia coli heat-labile enterotoxin B subunit (LT-B) between the first limited enzyme cutting section Nde I and the polyclonal section Noc I after the RBS of the pET32a, namely pET-LTB. Using this carrier can not only realize the prokaryotic secretion expression of the LT-B, but also can be expressed combining with its protective antigen protein.

Description

technical field [0001] The present invention relates to a carrier for prokaryotic expression of E. coli heat-labile enterotoxin B subunit (E. coli heat-labile enterotoxin B subunit, LT-B), in particular to a carrier for prokaryotic secretion and fusion expression of LT-B . Background technique [0002] Escherichia coli heat-labile enterotoxin (LT) consists of two subunits, A and B, among which the non-toxic B subunit (LT-B) has strong immunogenicity, especially for Strong mucosal immune activity. Therefore, when no target gene is inserted in the multi-cloning site region behind LT-B, the expressed protein can produce antibodies against Escherichia coli heat-sensitive enterotoxin, so it can be used as a vaccine component to prevent "tourist's diarrhea" and young animal diarrhea . [0003] At present, the prokaryotic expression of LT-B is mostly in the form of inclusion bodies, which brings difficulties to the purification of the expression product (target protein LT-B). I...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/66
Inventor 魏东芝马兴元王天文郑文云
Owner EAST CHINA UNIV OF SCI & TECH