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Soluble expression recombinant bacteria of K88 fimbria protein FaeG and fermental cultivation method thereof

A culture method and an adhesin technology, applied in the field of molecular biology, to achieve the effects of reducing the expression of inclusion bodies, high product purity and activity, and high consistency

Inactive Publication Date: 2016-11-16
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using fusion tags can effectively increase the soluble expression level of proteins, but to obtain mature target proteins, issues such as subsequent tag removal, protein recovery, and specific cleavage enzymes need to be considered

Method used

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  • Soluble expression recombinant bacteria of K88 fimbria protein FaeG and fermental cultivation method thereof
  • Soluble expression recombinant bacteria of K88 fimbria protein FaeG and fermental cultivation method thereof
  • Soluble expression recombinant bacteria of K88 fimbria protein FaeG and fermental cultivation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of recombinant bacteria co-expressed with adhesin protein FaeG and molecular chaperone GroEL / GroES

[0052] 1. The bacterial strains and plasmid vectors used in this example are shown in Table 1 below.

[0053] Table 1 Strains and plasmids

[0054]

[0055] 2. Main reagents and kits

[0056] PCR-related reagents: Taq DNA polymerase, dNTP (2.5mM), 10×Taq buffer, DNA marker (2kb and 10kb): Beijing Quanshijin Biotechnology Co., Ltd.;

[0057] Restriction enzyme: Dalian Bao Biological Engineering Co., Ltd.;

[0058] Agarose: Biowest Agarose aliquots;

[0059] Tryptone, yeast extract: British OXOID;

[0060] DNA gel recovery kit: OMEGA;

[0061] Plasmid extraction kit: OMEGA.

[0062] 3. Solution preparation

[0063] 3.1 Bacterial DNA extraction related solutions

[0064] PBS (0.012M): Weigh NaCl 8g, KCl 0.2g, NaCl 2 HPO 4 12H 2 O 3.58g, KH 2 PO 4 0.27g, add ddH 2 Dilute to 1L with O, and sterilize in aliquots at 121°C for 30 minutes...

Embodiment 2

[0153] Example 2: Expression, purification and primary and secondary structure identification of the adhesin protein FaeG

[0154] 1. Solution preparation

[0155] Reagents related to protein purification

[0156] (1) Buffer A: 25mmol / L Tris 3.0285g, 500mmol / L NaCl 29.25g, add ddH 2 Dilute O to 1L, and store by filtering with a 0.45um membrane filter.

[0157] (2) Buffer B: 25mmol / L Tris 3.0285g, 500mmol / L NaCl 29.25g, 500mmol / L imidazole 34.04g, add ddH 2 Dilute O to 1L, and store by filtering with a 0.45um membrane filter.

[0158] (3) Wash Buffer: 20% ethanol, 100mL absolute ethanol dissolved in 400mL ddH 2 O, mix well, and filter with a 0.45um membrane filter for storage.

[0159] 2. Purification of protein by nickel affinity chromatography

[0160] (1) Bacteria collection, the cells induced to express were centrifuged at 4°C, 10000g, for 5 minutes to collect the cells.

[0161] (2) Washing the bacteria, washing the bacteria three times with PBS, and resuspending th...

Embodiment 3

[0175] Embodiment 3: Optimization of fermentation conditions on the shake flask level of recombinant engineering bacteria

[0176] 1. Reagents

[0177] (1) Reference Example 1 for the preparation of LB medium, Kan, Amp, Cm, and IPTG.

[0178] (2) SDS-PAGE electrophoresis related solution reference example 1 (.

[0179] (3) TB medium (L): glycerol 5g; yeast powder 24g; peptone 12g; KH 2 PO 4 17 mmol; K HPO 4 72mmol.

[0180] (4) PBS (50mmol / L): Weigh NaCl 8g; KCl 0.2g; NaCl 2 HPO 4 12H 2 O 17.9 g; KH 2 PO 4 1.35g, add ddH 2 Dilute the volume to 1L with O, and sterilize at 121°C for 30min.

[0181] 2. SDS-PAGE detection of expression products

[0182] Shake flask fermentation, take 9mL fermentation broth, 4°C, 10000r / min centrifugation for 10min to collect the bacteria, ddH 2 O Wash the cells 3 times, resuspend the cells in 900mL 50mmol / L PBS (10-fold protein concentration), and ultrasonically break at 125w for 3min (the samples were fixed in a mixture of ice and w...

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Abstract

The invention discloses soluble expression recombinant bacteria of ETEC (Enterotoxigenic E. Coli) K88 fimbria protein FaeG. The soluble expression recombinant bacteria are characterized by firstly constructing a recombinant plasmid pET28a-FaeG for expressing an ETEC K88 fimbria FaeG gene, and then co-transforming the recombinant plasmid pET28a-FaeG and a carrier plasmid pGro7 of a molecular chaperone GroEL / GroES into a competence of Escherichia coli BL21 (DE3), thus obtaining the recombinant bacteria for co-expressing the fimbria FaeG gene and a molecular chaperone GroEL / GroES gene. The invention also discloses a fermental cultivation method of the recombinant bacteria. The recombinant bacteria provided by the invention have a higher soluble expression level of the fimbria protein FaeG, the expression quantity of an inclusion body is obviously reduced, an immunogen with a relative high purity can be obtained, and obtained protein has higher consistency with theoretic protein.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a soluble expression recombinant bacterium of enterotoxigenic Escherichia coli K88 adhesin protein FaeG, and also relates to a high-density fermentation and cultivation method of the recombinant bacterium. technical background [0002] Enterotoxigenic Escherichia coli (Enterotoxigenic E.coli, ETEC) can cause pullorum, yellow scour and swine edema in piglets. Infection with ETEC leads to diarrhea in piglets, which accounts for 30%-50% of the diarrhea rate in piglets. It is an important cause of piglet morbidity. Enteropathogens, and K88 is one of the most important ETEC pathogens causing diarrhea in piglets (Do et al 2005). [0003] The colonization of ETEC depends on the specific pili on the surface of the ETEC, which are called adhesins or colonization factor antigens (CFAs). Pili adhesin is composed of protein and has strong immunogenicity. The use of adhe...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/31C12P21/02C12R1/19
CPCC07K14/245C12P21/02
Inventor 彭健刘文亭周忠新王俊魏宏逵蒋思文
Owner HUAZHONG AGRI UNIV