Recombinant strain for expression of enterotoxin colibacillus adhesin gene and its application in vitelline antibody fodder

A technology of Escherichia coli and egg yolk antibody, which is applied in the fields of animal nutrition and feed science, can solve the problems of no expression, inappropriate large-scale production of egg yolk antibody, weak expression of fimbriae in vitro, etc.

Inactive Publication Date: 2008-01-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the above data, the method of directly separating and purifying pilin protein from ETEC strains is mainly used to prepare pili protein antigens. Since the expression of pili in general ETEC is very limited, and some pili are weakly or even not expressed in vitro, the method of separation and purification The amount of pili antigen obtained is small, the cost is high, and it is not suitable for the large-scale production of egg yolk antibody

Method used

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  • Recombinant strain for expression of enterotoxin colibacillus adhesin gene and its application in vitelline antibody fodder
  • Recombinant strain for expression of enterotoxin colibacillus adhesin gene and its application in vitelline antibody fodder
  • Recombinant strain for expression of enterotoxin colibacillus adhesin gene and its application in vitelline antibody fodder

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of recombinant plasmid pET8818 expressing K88ac adhesin gene faeG and F18ac adhesin gene fadF gene

[0032] 1 Materials and methods

[0033] 1.1 Strains and plasmid vectors

[0034] Escherichia coli K88ac standard strain C83715 was purchased from China Veterinary Drug Control Institute. Escherichia coli F18ac strain 2134P was kindly donated by Professor Cheng Darong from Veterinary Hospital of Yangzhou University.

[0035] Escherichia coli DH5α, Escherichia coli BL21(DE3) and plasmid vector pET-28a were purchased from Novagen.

[0036] The cloning vector pMD18-T was purchased from Takara.

[0037] 1.2 Medium

[0038] LB Broth (LB Broth): tryptone 10g, yeast extract 5g, NaCl 10g, distilled water to 1000mL, adjust pH to pH7.0, 121°C 1.05kg / cm 2 Sterilize by high pressure steam for 20min, store at room temperature for later use.

[0039] LB Nutrient Agar (LB Nutrient Agar): tryptone 10g, yeast extract 5g, NaCl 10g, distilled water to 1000mL, a...

Embodiment 2

[0060] Example 2: Induced expression and extraction and purification of FaeG-FedF fusion protein

[0061] 1 Materials and methods

[0062] 1.1 Reagents

[0063] Tryptone, yeast extract, agar powder, isopropyl-β-D-thiogalactopyranoside (lsopropyl-D-1-thiogalactopyranoside, IPTG), β-mercaptoethanol, sodium lauryl sarcosinate (referred to as SKL), polyethylene glycol 4000 (referred to as PEG4000), oxidized glutathione, and reduced glutathione are products of Shanghai Sangong Bioengineering Co., Ltd.

[0064] 1.2 Antibody

[0065] The mouse anti-His·Tag antibody was the product of Novagen Company, and the goat anti-mouse IgG labeled with horseradish peroxidase was purchased from SBA Company.

[0066] 1.3 Buffer

[0067] BufferA: 50mol / LTris, 0.5mol / LEDTA, 50mol / LNaCl, 5% glycerol, adjust the pH to pH8.0, add 0.5mmol / L dithiothreitol when used.

[0068] TE (pH8.0): 10mmol / L Tris·Cl, 1mmol / L EDTA.

[0069] PBS (pH7.4): PBS: Weigh 8gNaCl, 0.2gKCl, 1.44gNa 2 HPO 4 , 0.24g KH ...

Embodiment 3

[0081] Example 3: Preparation of Egg Yolk Antibody Using FaeG-FedF Fusion Protein as Immunogen

[0082] 1 Materials and methods

[0083] 1.1 Adjuvants

[0084] White oil adjuvant: 94mL white oil, 6mL Siben-80, 2g aluminum stearate, after mixing evenly, 121°C 1.05kg / cm 2 Autoclaved for 30min, cooled to room temperature for later use.

[0085] 1.2 Antibody

[0086] K88ac-positive serum antibody was prepared by the Experimental Animal Center of Wuhan Institute of Virology, Chinese Academy of Sciences, F18 "a" single-factor serum was donated by Professor Cheng Darong from the Veterinary Hospital of Yangzhou University, horseradish peroxidase-labeled mouse anti-chicken IgG was purchased from Sigma

[0087] 1.3 Buffer

[0088] PBSI buffer: NaCl10.0g; KH 2 PO 4 0.25g; Na 2 HPO 4 12H 2O3.58g; KCL0.25g, dissolved in 900mL distilled water, adjusted pH to 7.2 with 1mol / L HCl, adjusted to 1000mL with distilled water, autoclaved at 121°C for 15min for later use

[0089] PBSII buf...

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Abstract

The invention pertains to the scientific technical field of animal nutrition and feed, meanwhile, relates to application of animal molecular biologic technology. Particularly, the invention relates to an expression for constructing escherichia coli K88 of enterotoxin of chitterlings and recomposed escherichia coli of F18 adhesion gene faeG and fedF and application thereof in preparation of egg-yolk antibody feed. The escherichia coli expressed the recomposed plasmids of escherichia coli K88ac of the enterotoxin and the adhesion genes faeG, F18ac and fedF is constructed by constructing fusion gene of faeG and fedF by using connecting peptid, and inserting the fusion gene into a prokaryotic expression carrier pET28a. The recomposed escherichia coli BL21 (DE3) / pET-8818 of the invention are preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M207090. The invention also discloses application of the recomposed escherichia coli with the preservation number of CCTCC NO: M207090 in the preparation of the egg-yolk antibody feed.

Description

technical field [0001] The invention relates to the technical field of animal nutrition and feed science, and also relates to the application of animal molecular biology. Specifically, the present invention relates to a recombinant bacterium expressing porcine enterotoxigenic Escherichia coli K88 and F18 adhesin genes faeG and fedF and its application in preparing yolk antibody feed. Background technique [0002] In large-scale pig production, enterotoxigenic Escherichia coli (ETEC for short) is the main pathogenic factor causing diarrhea in newborn piglets and weaned piglets. K88 is the most important pili genotype in ETEC causing diarrhea in weaned piglets (Osek J et al. :265-70); among them, the K88ac subtype is the most common. In Denmark, Ojeniyi et al. (Ojeniyi B et al. Detection of fimbrial and toxin genes in and their prevalence inpiglets with diarrhea. The applfication of colony hybridization assay, polymerase chain reaction and phenotype assays. J Vet Med B, 1994...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/62A23K1/16A23K20/147
Inventor 彭健王劼蒋思文
Owner HUAZHONG AGRI UNIV
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