Recombinant Lactococcus lactis expressing and secreting single-chain antibody against porcine transmissible gastroenteritis virus and its preparation method

A technology of Lactococcus lactis and single-chain antibody, applied in the field of bioengineering, can solve problems such as unsatisfactory effects, and achieve the effect of reducing gastric acid and pepsin damage

Active Publication Date: 2022-05-17
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, vaccination is one of the ways to prevent the occurrence of this disease, but the effect of the developed PED inactivated and attenuated vaccines is not ideal

Method used

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  • Recombinant Lactococcus lactis expressing and secreting single-chain antibody against porcine transmissible gastroenteritis virus and its preparation method
  • Recombinant Lactococcus lactis expressing and secreting single-chain antibody against porcine transmissible gastroenteritis virus and its preparation method
  • Recombinant Lactococcus lactis expressing and secreting single-chain antibody against porcine transmissible gastroenteritis virus and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] This embodiment relates to a method for constructing and secreting recombinant Lactococcus lactis expressing a single-chain antibody against porcine transmissible gastroenteritis virus, specifically comprising:

[0028] ① Design the sequence with the secretion signal peptide USP45, the secretion enhancing signal peptide LEISSTCDA and the thioredoxin A gene (trxA), add the Nco I restriction site at the 5' end, add EcoR I at the 3' end, and Add Kpn I at both ends of the trxA gene to obtain the Seq ID No.1 sequence; use CodonOptimWiz software to optimize the codons of the anti-porcine transmissible gastroenteritis virus single-chain antibody TZZ 19 sequence, add EcoR I at the 5' end, and Add 6×His sequence and Hind III to the 3' end to obtain Seq ID No.2 sequence. The designed Seq ID No.1 and Seq ID No.2 sequences were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0029] ②The synthetic Seq ID No.1 sequence and the synthetic Seq ID No.2 sequence were sequential...

Embodiment 2

[0032] This example relates to the identification of anti-porcine transmissible gastroenteritis virus single-chain antibody secreted by recombinant Lactococcus lactis, specifically including:

[0033] i) Inoculate the overnight cultured recombinant Lactococcus lactis into two tubes of 10 mL SGM17B medium with chloramphenicol at a ratio of 1:25, and cultivate until the OD600 reaches 0.4. Nisin with a final concentration of 10ng / mL was added to one of the tubes, and the culture was continued for 5-8h.

[0034] ii) Extracting the single-chain antibody secreted by the recombinant Lactococcus lactis, 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 30min; add acetone, 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. T...

Embodiment 3

[0038] This example relates to the neutralization effect of the single-chain antibody secreted by recombinant Lactococcus lactis on transmissible gastroenteritis virus. The neutralization effect, the method is as follows:

[0039] The single-chain antibody TZZ 19 purified from recombinant Lactococcus lactis was serially diluted twice, and the single-chain antibody TZZ 19 of each dilution was mixed with an equal volume of 1000 TCID50 / mL TGEV, and the mixture was incubated for 1 h. The single-chain antibody ZW 88 that does not react with TGEV stored in our laboratory was used as a negative control. After 1 hour, the TGEV-single-chain antibody TZZ 19 mixture was added to the 24-well plate and incubated at 37° C. for 1 hour. The mixed solution in the 24-well plate was replaced with DMEM solid medium containing 1% agar, and cultured for 36-48h. Using crystal violet staining, the number of plaques was recorded, and the plaque reduction rate was calculated: [1-(average number of pl...

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Abstract

A recombinant Lactococcus lactis expressing and secreting anti-porcine transmissible gastroenteritis virus single-chain antibody and its preparation method. A fusion sequence, the fusion sequence has the amino acid sequence shown in SEQ ID No.4. The single-chain antibody secreted by the recombinant Lactococcus lactis can specifically bind to the porcine transmissible gastroenteritis virus, and can be used to block the infection of the porcine transmissible gastroenteritis virus and provide protection for piglets infected with the virus.

Description

technical field [0001] The invention relates to a technology in the field of bioengineering, in particular to a recombinant Lactococcus lactis expressing and secreting a single-chain antibody against porcine transmissible gastroenteritis virus and a preparation method thereof. Background technique [0002] Transmissible gastroenteritis virus (TGEV) causes pig transmissible gastroenteritis (transmissible gastroenteritis, TGE). Once a pig in a pig farm is infected with TGEV, pigs of all ages will be infected soon. At present, vaccination is one of the ways to prevent the occurrence of this disease, but the effect of the inactivated and attenuated PED vaccines that have been developed is not ideal. Contents of the invention [0003] The present invention aims at the above-mentioned deficiencies in the prior art, and proposes a recombinant Lactococcus lactis expressing and secreting a single-chain antibody against porcine transmissible gastroenteritis virus and a preparation m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/13C12N15/74A61K35/744A61P1/00C12R1/01
CPCC07K16/10C12N15/746A61K35/744A61P1/00Y02A50/30
Inventor 朱建国张凡庆王凤青张蕾闫洁新程曼玲
Owner SHANGHAI JIAOTONG UNIV
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