Eukaryon expression of coli heat-sensitive toxin B subunit

An expression method, Escherichia coli technology, applied in the direction of botanical equipment and methods, biochemical equipment and methods, applications, etc., to achieve the effects of easy detection and purification, simple and fast specificity, and less interference factors

Inactive Publication Date: 2007-07-04
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

[0004] Escherichia coli thermosensitive toxin B subunit (LT-B) with mucosal immune activity has been expressed in a variety of ways, such as expression methods such as Escherichia coli, Bacillus subtilis or transgenic tomato (1. Clements, J.D., Hartzog, N.M. , Lyon, F.L., 1988.Adjuvant activity of Escherichia coli heat-labile enterotoxin and effect on the induction of oral tolerance in mice to unrelated protein antigens.Vaccine 6, 269., 2.Wang J, Li L, Zheng J, et al. Highly efficient expression, purification of recombinant LTB protein and its activity against mucosal immunoadjuvant by nasal immunization. Chin Med J (Engl). 2003 Jul; 116 (7): 1115-1117., 3. Walmsley AM, Alvarez ML, Jin Y.et al.Expression of the B subunit of Escherichia coliheat-labile enterotoxin as a fusion protein in transgenic tomato.Plant Cell Rep.2003 Jun; 21(10):1020-1026.), but whether in protein expression, post-transcriptional processing In terms of high-level structure formation, there are still varying degrees of defects in protein purification and detection

Method used

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  • Eukaryon expression of coli heat-sensitive toxin B subunit
  • Eukaryon expression of coli heat-sensitive toxin B subunit
  • Eukaryon expression of coli heat-sensitive toxin B subunit

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Experimental program
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Effect test

Embodiment

[0027] 1. Cloning and sequencing of LT-B gene with signal peptide sequence

[0028] Cloned from pathogenic Escherichia coli E.coli O6:H16 (LT) inserted into the pGEM-T vector + , ST + ) LTB whole gene as a template, using primers Pb1: 5'-ATG AAT AAA GTA AAA TGT TAT-3 and Pb2: 5'-GAA TTC TAG TTT TCCATACTGATTGCC-3, the LT with signal peptide sequence was amplified by high-fidelity PCR -B (sequence attached below), subcloned into pGEM-T, LT-B with signal peptide sequence was correct after sequencing, its sequence is as follows, a total of 372bp, which can encode 103 amino acids of mature peptide LT-B.

[0029] 1 atgaataaag taaaatgtta tgttttatt acggcgttac tatcctctct atgtgcatac

[0030] 71 ggagctcccc agtctattac agaactatgt tcggaatatc gcaacacaca aatatatacg

[0031] 121 ataaatgaca agatactatc atatacggaa tcgatggcag gtaaaagaga aatggttatc

[0032] 181 attacattta agagcggcgc aacatttcag gtcgaagtcc cgggcagtca acatatagac

[0033] 241 tcccaaaaaa aagccattga aaggatgaag gacacattaa gaatcacata ...

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Abstract

An eucaryotic expression of E.coli heat-labile enterotoxin B subunit, LT-B is prepared by defining nucleic acid endoenzyme site, connecting LT-B nucleic acid sequence with pPICZ alpha A to obtain recombinant expression carrier, inducing recombinant expression carrier into Pichiapastoris X-33 or KM71, and fermentation expressing to obtain target protein LT-B. It has high economical value.

Description

technical field [0001] The invention relates to a eukaryotic expression method of E. coli heat-labile enterotoxin B subunit (LT-B). Background technique [0002] As early as the 1940s, it was discovered that certain serotypes of Escherichia coli (E.coli) could cause epidemic diarrhea in young animals and infants, and realized that not all E.coli had a "normal" intestinal tract The E.coli that can cause diarrhea in humans and animals is called enteropathogenic E.coli (Enteropathogenic E.coli, EPEC). Further studies have shown that the main reason for EPEC to cause diarrhea in humans and animals is the production of enterotoxins. Therefore, this E.coli that can produce enterotoxins is also called enterotoxigenic E.coli (Enterotoxigenic E.coli, ETEC) . [0003] ETEC mainly produces two types of enterotoxins: one is E.coli heat-stable enterotoxin (ST), and the other is E.coli heat-labile enterotoxin (LT) . LT molecule is a six-mer toxin protein composed of one A subunit (LT-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/64C12P21/02C12N15/81
Inventor 魏东芝马兴元郑文云王天文罗晨马昱澍
Owner EAST CHINA UNIV OF SCI & TECH
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