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Recombinant lactococcus lactis for secretory expression of swine-derived anti-enterotoxigenic escherichia coli K88 pilus single-chain antibody and preparation method of recombinant lactococcus lactis

A technology of Lactococcus lactis and Escherichia coli, applied in the direction of virus/bacteriophage, anti-bacterial immunoglobulin, microbial-based methods, etc., can solve the problem of single-chain antibody destruction

Active Publication Date: 2020-12-01
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a recombinant Lactococcus lactis that secretes and expresses pig-derived anti-enterotoxigenic E.

Method used

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  • Recombinant lactococcus lactis for secretory expression of swine-derived anti-enterotoxigenic escherichia coli K88 pilus single-chain antibody and preparation method of recombinant lactococcus lactis
  • Recombinant lactococcus lactis for secretory expression of swine-derived anti-enterotoxigenic escherichia coli K88 pilus single-chain antibody and preparation method of recombinant lactococcus lactis
  • Recombinant lactococcus lactis for secretory expression of swine-derived anti-enterotoxigenic escherichia coli K88 pilus single-chain antibody and preparation method of recombinant lactococcus lactis

Examples

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Effect test

Embodiment 1

[0038] Example 1 Construction of recombinant Lactococcus lactis secreting and expressing single-chain antibody against enterotoxigenic Escherichia coli K88 pili

[0039] Design the sequence with secretion signal peptide USP45, secretion enhancing signal peptide LEISSTCDA and thioredoxin A gene (trxA), add Nco I restriction site at the 5' end, add EcoR I at the 3' end, and Add Kpn I to both ends of the trxA gene; use Codon OptimWiz software to optimize the codons of the EZZ 3 sequence of the anti-enterotoxigenic E. and Hind III. The designed sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0040]The sequence of the secretion signal peptide USP45, the secretion enhancement signal peptide LEISSTCDA and the thioredoxin A gene (trxA), and the sequence of the codon-optimized single-chain antibody EZZ 3 were sequentially connected into pNZ8148, and the MC1061F-competent cells were transformed to obtain Recombinant plasmid pNZ8148-USP45-LEISSTCDA-trxA-EZZ 3 (ab...

Embodiment 2

[0042] Example 2 Identification of recombinant lactic acid bacteria secreting anti-enterotoxigenic Escherichia coli K88 pili single-chain antibody

[0043] The recombinant lactic acid bacteria cultivated overnight were inoculated into two tubes of 10 mL SGM17B medium with chloramphenicol at a ratio of 1:25, and cultivated until the OD600 reached 0.4. Nisin (nisin, also known as nisin) with a final concentration of 10ng / mL was added to one of the tubes, and the culture was continued for 5-8h.

[0044] Extract the single-chain antibody secreted by the recombinant lactic acid bacteria, the method is: centrifuge the induced bacterial liquid, collect the culture supernatant; add 1% (v / v) 2% sodium deoxycholate to the supernatant, let it stand for 30min; add acetone , let stand at 4°C for 2h; centrifuge at 12,000×g for 30min, wash the precipitate twice with pre-cooled acetone, and centrifuge to collect the precipitate; suspend the precipitate in protein loading buffer or PBS. The s...

Embodiment 3

[0047] Example 3 Inhibition of adhesion of single-chain antibodies secreted by recombinant lactic acid bacteria to enterotoxigenic Escherichia coli

[0048] The method of adhesion inhibition experiment is as follows: the monolayer IPEC-J2 cells are used for the adhesion inhibition experiment, and bacteria (MOI=50:1) are mixed with 50 μg / mL or 5 μg / mL single-chain antibody purified from recombinant Lactococcus lactis EZZ 3 was pre-acted for 30 minutes, and then the ETEC-single-chain antibody EZZ 3 mixture was added to IPEC-J2 cells and reacted at 37°C for 1 hour. The 12-well plate was incubated at 37°C for 1 h, and washed 5 times with PBS after 1 h to remove unadhered ETEC. Add 0.5% Triton X-100 to the 12-well plate, and repeatedly pipette to lyse the cells. The lysate was centrifuged at 8,000×g for 10 min, and the precipitate was serially diluted 10 times, and then spread on an LB plate to count the number of colonies. The mouse anti-K88ac pilus polyclonal antibody was used ...

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Abstract

The invention relates to recombinant lactococcus lactis for secretory expression of a swine-derived anti-enterotoxigenic escherichia coli K88 pilus single-chain antibody and a preparation method of the recombinant lactococcus lactis, and belongs to the technical field of genetic engineering. The recombinant lactococcus lactis carries a fusion sequence for secretory and expression of an anti-enterotoxigenic escherichia coli K88 pilus single-chain antibody. The fusion sequence has a sequence as shown in SEQ ID No. 1 and a single-chain antibody sequence as shown in SEQ ID No. 2, wherein the sequence as shown in SEQ ID No. 1 is a sequence containing a secretion signal peptide USP45, a secretion enhancing signal peptide LEISSTCDA and a thioredoxin gene. The molecular weight of the obtained secreted single-chain antibody for resisting enterotoxigenic escherichia coli K88 pilus is about 45kDa, and the secreted single-chain antibody can specifically recognize enterotoxigenic escherichia coli,block bacterial infection and protect piglets against enterotoxigenic escherichia coli diseases.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a pig-derived single-chain antibody against enterotoxigenic E. Preparation method of recombinant Lactococcus lactis for enterotoxigenic Escherichia coli K88 pilus single-chain antibody, gene encoding the gene for secreting and expressing anti-enterotoxigenic Escherichia coli K88 pilus single-chain antibody, expression vector for secreting and expressing anti-enterotoxigenic Escherichia coli K88 pilus single-chain antibody and host cells. Background technique [0002] Escherichia coli was first isolated from feces by Theodore Escherich in 1885. To commemorate his research, Escherichia coli was named after him. Some Escherichia coli are components of normal intestinal flora, others cause diarrhea, and the most common pathogenic Escherichia coli is enterotoxigenic Escherichia coli (ETEC). The main virulence factors of enterotoxigenic E. coli are adhesin and enterotoxin. ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/62C12N15/74C07K19/00C07K16/12C12R1/46
CPCC07K16/1232C12N15/74C12N2800/22C07K2319/02C07K2319/35Y02A50/30
Inventor 朱建国张凡庆王凤青张蕾闫洁新程曼玲
Owner SHANGHAI JIAO TONG UNIV
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