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A kind of human source enterotoxigenic Escherichia coli adhesin etpa fusion protein and application thereof

A technology of Escherichia coli and fusion protein, applied in the direction of anti-bacterial immunoglobulin, egg-derived immunoglobulin, bacteria, etc., can solve the problem of no cross-protection effect of heterologous pili, and achieve good antibody effect and high titer Effect

Active Publication Date: 2018-06-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, more than 22 CFAs have been identified in human ETEC, the common ones are CFA / I, CS1-CS7, CS14, CS17, CS21 (Qadri et al2005), but there is no cross-protection between heterologous pili (Qadri et al2006)

Method used

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  • A kind of human source enterotoxigenic Escherichia coli adhesin etpa fusion protein and application thereof
  • A kind of human source enterotoxigenic Escherichia coli adhesin etpa fusion protein and application thereof
  • A kind of human source enterotoxigenic Escherichia coli adhesin etpa fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Construction of Enterotoxigenic Escherichia coli EtpA Recombinant Plasmid

[0050] 1. The bacterial strains and plasmid vectors used in this example are shown in Table 1 below.

[0051] Table 1 Strains and plasmids

[0052]

[0053]

[0054] 2. Main reagents and kits

[0055] PCR-related reagents: Taq DNA polymerase, dNTP (2.5mM), 10×Taq buffer, DNA marker (2kb and 10kb): Beijing Quanshijin Biotechnology Co., Ltd.;

[0056] Restriction enzyme: Dalian Bao Biological Engineering Co., Ltd.;

[0057] Agarose: Biowest Agarose aliquots;

[0058] Tryptone, yeast extract: British OXOID;

[0059] Trypsin digested soybean broth: American Bidi Medical Devices Co., Ltd.;

[0060] DNA gel recovery kit: Shanghai Sangon Biotechnology Co., Ltd.;

[0061] Plasmid extraction kit: Shanghai Sangon Biotechnology Co., Ltd.

[0062] 3. Solution preparation

[0063] 3.1 Bacterial DNA extraction related solutions

[0064] PBS (0.012M): Weigh NaCl8g, KCl0.2g, NaCl 2 HP...

Embodiment 2

[0121] Induced expression and extraction and purification of recombinant proteins

[0122] 1. Reagents

[0123] Tryptone, yeast extract: British OXOID;

[0124] Trypsin digested soybean broth: American Bidi Medical Devices Co., Ltd.;

[0125] Tris base, glycine, SDS: Amresco aliquots;

[0126] IPTG, Amp, Kan, oxidized glutathione, reduced glutathione, PEG4000: BIOSHARP packaging;

[0127] 2. Solution preparation

[0128] LB medium was as shown in Example 1.

[0129] IPTG stock solution (1mol / L): add 1g of powder to 4.2mL sterilized ddH on the aseptic operating table 2 O, filtered through a sterile filter and dispensed into 1.5mLEP tubes.

[0130] SDS-PAGE electrophoresis related solutions:

[0131] 1) 2× protein sample solution: Tris-HCl (1M, pH=6.8) 10mL, SDS 4g, bromophenol blue 200mg, glycerol 20mL, add ddH 2 Dilute O to 100mL, and add 2-3% β-mercaptoethanol (ready to use) when using.

[0132] 2) 5× electrophoresis buffer: Tris15.10g, Gly72.06g, SDS5g, add ddH 2 O...

Embodiment 3

[0161] Immunization of laying hens with recombinant protein to prepare egg yolk antibody

[0162] 1. Reagents

[0163] White oil, aluminum hardate, Siben-80, Tween-80: gifts from Wuhan Keqian Biological Products Co., Ltd.

[0164] HRP rabbit anti-chicken IgG: sigma

[0165] Chromogenic solution Supersignal west pico test kit: Thermo

[0166] 2. Solution preparation

[0167] ELISA related reagents:

[0168] PBS (0.012M): Weigh NaCl8g, KCl0.2g, NaCl 2 HPO 4 12H 2 O3.58g, KH 2 PO 4 0.27g, add ddH 2 Dilute to 1L with O, and sterilize in aliquots at 121°C for 30 minutes.

[0169] Coating solution (0.05M carbonate buffer): NaHCO 3 2.93g, Na 2 CO 3 1.59g, add ddH 2 After O900mL is completely dissolved, adjust the pH to 9.6, dilute to 1L, and place at room temperature.

[0170] Washing solution: Tween-20 with a final concentration of 0.05% was added to PBS.

[0171] Blocking solution: 3% bovine serum albumin (BSA) dissolved in washing solution.

[0172] OPD chromogeni...

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Abstract

The invention discloses a fusion protein of human enterotoxin E. coli adhesin EtpA and its application. The invention provides a recombinant plasmid pGEX‑etpA containing the adhesin gene of human enterotoxin E. coli. The plasmid was transformed into Escherichia coli BL21(DE3), and the recombinant expression strain BL21(DE3) / pGEX‑etpA was induced to express the fusion protein EtpA. The expressed recombinant protein was used to actively immunize first-laying hens, and the yolk antibody against this protein was prepared. The extracted yolk antibody verified that the recombinant protein had good antigenicity, and the mouse intestinal challenge test and in vitro experiments proved that The purified egg yolk antibody can inhibit the adhesion of ETEC strain to epithelial cells in mouse intestine and in vitro.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a fusion protein of human enterotoxigenic Escherichia coli adhesin EtpA and its application, by constructing the adhesin gene etpA recombinant plasmid pGEX-etpA of human enterotoxigenic Escherichia coli , transform it into E. coli to express the fusion protein, and immunize laying hens. The purified egg yolk antibody can inhibit the adhesion of ETEC strains to epithelial cells in the intestinal tract of mice and in vitro. Background technique [0002] Enterotoxigenic Escherichia coli (Enterotoxigenic Escherichia coli, ETEC) is the main pathogenic bacteria causing diarrhea (Black1990; Ericsson2003). According to statistics, ETEC can cause 280-400 million times of diarrhea in children under 5 years old every year. The severity of the disease can range from mild diarrhea to severe cholera-like, resulting in about 300,000-500,000 infant deaths per year (WHO2004) ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C07K19/00C07K16/12C07K16/02A61K39/40A61P31/04A61P1/12C12P21/02C07K1/113
Inventor 彭健王荣蒋思文潘中勉
Owner HUAZHONG AGRI UNIV
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