A kind of method for preparing tetramer of Asian I type foot-and-mouth disease virus polypeptide

A foot-and-mouth disease virus, tetramer technology, applied in the direction of viral peptides, viruses/phages, biochemical equipment and methods, etc.

Active Publication Date: 2022-05-27
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, the Tetramer technology has not been applied to the identification of the function of the foot-and-mouth disease virus polypeptide bound by the porcine SLA class I molecule in China.

Method used

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  • A kind of method for preparing tetramer of Asian I type foot-and-mouth disease virus polypeptide
  • A kind of method for preparing tetramer of Asian I type foot-and-mouth disease virus polypeptide
  • A kind of method for preparing tetramer of Asian I type foot-and-mouth disease virus polypeptide

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Embodiment Construction

[0062] Now further illustrate the present invention in conjunction with embodiment.

[0063] First, the reagents involved in the present invention are described;

[0064] IPTG, DNA Marker and Protein Molecular Weight Marker are all products of TaKaRa Company; Propionamide, PBS solution, NH 4 Cl, DMSO, Tris-base, L-arginine, EDTA, reduced glutathione (GSH), oxidized glutathione (GSSG), PMSF, ammonium persulfate, TEMED, DTT, Coomassie brilliant blue R250 Purchased from Sangon Bioengineering Co., Ltd.; BCA protein concentration assay kit, western blot developer and fixer were purchased from Beyontian; protein concentration ultrafiltration tubes were purchased from MilliPore; Anti-His Tag Mouse Monoclonal Antibody and HRPConjugated Goat anti -Mouse IgG was purchased from Ametek; ELISpot PLUSfor Porcine IFN-γ Kit was purchased from Mabtech Company; Porcine Peripheral Blood Lymphocyte Separation Kit was purchased from TBD Company; Con-A (concanavalin A) was purchased from SIGMA C...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a tetramer of Asian type I foot-and-mouth disease virus polypeptide. For the designed foot-and-mouth disease virus polypeptide, the present invention will adopt two methods of ELISPOT and construction of tetramer to screen and identify the foot-and-mouth disease virus polypeptide, construct the heavy chain tetramer of purse pig SLA-2, and use it in the prokaryotic expression system pET-21a(+) / BL21 was expressed and inclusion bodies were extracted; foot-and-mouth disease virus polypeptides were screened for polypeptides that could cause specific CTL reactions by ELISPOT method, and then the polypeptides were refolded and biotinylated with the heavy chain of SLA‑2 and the existing light chain β2m And react with FITC-labeled streptavidin to generate SLA-2-BSP / peptide tetramer, and then detect the function of tetramer by flow cytometry. The present invention has identified a specific polypeptide epitope of porcine Asian type I foot-and-mouth disease virus VP1 through the ELISPOT method, successfully constructed a peptide tetramer, detected its specific CTL, and preliminarily established a porcine tetramer technology platform for To lay the foundation for the study of pig-specific CTL immune response and T epitope screening.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a tetramer of Asian type I foot-and-mouth disease virus polypeptide. Background technique [0002] Foot-and-mouth disease is an acute thermal and highly contagious infectious disease caused by Foot and Mouth Disease Virus (FMDV). It mainly affects artiodactyls. Pigs are a relatively susceptible group. Blisters and festers are the main features, and are the most contagious among livestock diseases. Every outbreak of foot-and-mouth disease will bring great losses to the agricultural economy. Therefore, the International Office of Epizootics (OIE) has always listed it as the top of the list of Class A animal diseases that must be reported. Therefore, the prevention and diagnosis of FMD in pigs is particularly important. At present, the prevention and treatment of FMD virus mainly relies on traditional inactivated vaccines. Inactivated vaccines have ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/09
CPCC07K14/005C12N2770/32122
Inventor 高凤山
Owner DALIAN UNIV
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