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Single-chain antibody of swine-origin enterotoxigenic Escherichia coli K88 FaeG resistant protein and preparation method of single-chain antibody

A single-chain antibody and Escherichia coli technology, applied in the field of genetic engineering, can solve problems such as the unsatisfactory effect of antibiotics on bacterial infection

Active Publication Date: 2016-10-12
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the problem of bacterial drug resistance is becoming more and more serious, and the effect of antibiotics on bacterial infections is not ideal

Method used

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  • Single-chain antibody of swine-origin enterotoxigenic Escherichia coli K88 FaeG resistant protein and preparation method of single-chain antibody
  • Single-chain antibody of swine-origin enterotoxigenic Escherichia coli K88 FaeG resistant protein and preparation method of single-chain antibody
  • Single-chain antibody of swine-origin enterotoxigenic Escherichia coli K88 FaeG resistant protein and preparation method of single-chain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of the single-chain antibody of pig-derived anti-enterotoxigenic Escherichia coli K88FaeG protein

[0028] 1. The heavy chain variable region sequence (AF064686.1; AF064687.1; AF064688.1; AF064689.1; AF064690.1) and light chain variable region sequence (KF561242.1) of the porcine antibody coding gene published by GenBank; GQ867594.1; GQ867595.1) designed primers for amplifying antibody light and heavy chains (Table 1), in which VH1F, VH1R and VH2R were used to amplify the VH region; VL1F, VL2F, VL3F and VL1R were used to amplify the VL region ; VH3F and VH3R are used to add restriction sites and Linker sequences to VH genes; VL4F, VL5F and VL6F are used to add restriction sites to VL genes amplified by VL1F, VL2F and VL3F, and VL2R is used to add Linker sequences to VL genes. Among them, VH3F contains a Sfi I restriction site, VL 2R contains a Not I restriction site; VH3R, VL4F, VL5F, and VL6F contain complementary Linker sequences (the restriction...

Embodiment 2

[0043] Example 2 Indirect ELISA screening of antigen-specific ScFv

[0044] Take the purified prokaryotically expressed ETEC K88FaeG protein (prepared in our laboratory), dilute it to 5 μg / mL with antigen coating solution (50 mmol / L sodium bicarbonate solution, pH=9.6), add it to a 96-well plate, 100 μL per well, Coat overnight at 4°C; add 200 μL of 5% skimmed milk powder solution to each well, block at 37°C for 1 hour, and wash 3 times with PBS; mix 50 μL of purified protein supernatant with 50 μL of 4% skim milk powder solution and add to the above wells, at 37°C Incubate for 2 h, wash with PBST 3 times; add 100 μL (1:2000) of E-Tag Mouse mAb (E-tag mouse monoclonal antibody purchased from RayBiotech Company), react at 37°C for 2 h, wash with PBST 3 times; add hydrogen peroxide to label Goat anti-mouse IgG secondary antibody (purchased from Invitrogen, USA) 1000 μL (1:4000), reacted at 37°C for 1 h, washed 3 times with PBST; developed color with TMB for 15 min, terminated re...

Embodiment 3

[0046] The obtained single-chain antibody coding gene was sequenced to prove that it consists of 762 nucleotides and 254 amino acids deduced accordingly. The nucleotide sequence is shown in SEQ ID No: 4, and the amino acid sequence is shown in SEQ ID No: 4. ID No: 3 shown.

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Abstract

The invention discloses a single-chain antibody of swine-origin enterotoxigenic Escherichia coli K88 FaeG resistant protein and a preparation method of the single-chain antibody. The single-chain antibody is formed by connecting a heavy-chain variable region VH and a light-chain variable region VL of an antibody through short connecting peptide, an amino acid sequence of the light-chain variable region VL is shown as SEQ ID No:1, and an amino acid sequence of the heavy-chain variable region is shown as SEQ ID No:2. Molecular weight of the single-chain antibody is about 28 kDa, the single-chain antibody can specifically recognizes enterotoxigenic Escherichia coli K88 and can be used for blocking infection and adhesion of the enterotoxigenic Escherichia coli K88.

Description

technical field [0001] The invention relates to a pig-derived single-chain antibody against enterotoxigenic Escherichia coli K88FaeG protein and a preparation method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Post-weaning diarrhea (PWD) occurs when piglets are weaned or in the first week after weaning. The main clinical symptoms are diarrhea, dehydration, slow growth or stop growth, which causes a large number of piglets to die. The pig industry has caused huge economic losses. Specific serotypes of enterotoxigenic E. coli (ETEC) are responsible for more than 50% of cases of diarrhea in weaned piglets. At present, the problem of bacterial drug resistance is becoming more and more serious, and the effect of antibiotics on bacterial infections is not satisfactory. Single-chain antibody is a kind of genetically engineered antibody. With its small molecular weight, high specificity, strong penetrating power, and easy modific...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13C12N15/70
CPCC07K16/005C07K16/1232C07K2317/622C12N15/70
Inventor 朱建国张凡庆王苗苗
Owner SHANGHAI JIAO TONG UNIV
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