犬降钙素原蛋白多克隆抗体制备及应用分析

doi:10.16431/j.cnki.1671-7236.2020.03.029

Abstract

Abstract: The aim of this study was to obtain high-purity canine procalcitonin recombinant protein and evaluate its clinical application.PCT gene was cloned into prokaryotic expression pET-30a(+) vector using the method of E.coli expressing foreign protein.After identified by enzyme digestion and sequencing,the recombinant plasmid was transferred into E.coli BL21(DE3) and induced by 1 mmol/L IPTG for protein.The recombinant protein was purified by nickel column,and polyclonal antibody was prepared by immunizing mice with purified protein.The antibody titer and specificity of procalcitonin were detected by indirect ELISA and Western blotting,respectively.The serum PCT in healthy and inflammatory dogs were detected by ELISA method.Enzyme digestion and sequencing tests confirmed canine PCT gene was successfully inserted into pET-30a(+).SDS-PAGE result showed that canine PCT protein was soluble expressed with a molecular weight of 14.9 ku.PCT antiserum titer was evaluated by indirect ELISA method and the best titer was 1:51 200.The specificity of polyantibody was checked by Western blotting,and the serum exhibited high affinity to PCT protein.The ELISA result showed the PCT in serum had positive correlation with the degree of inflammation.The results indicated that canine PCT protein polyclonal antibody was successfully prepared,and canine serum PCT protein was a potential new indicator for serious bacterial inflammation infection.

Key words: procalcitonin (PCT); prokaryotic expression; purification; polyclonal antibody preparation

CLC Number:

Q786

WANG Dong, LIU Ying, ZHANG Kang, ZHANG Hongyan, SU Feng, MA Weiming. Preparation and Application Analysis of Canine Procalcitonin Protein Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 892-901.

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Preparation and Application Analysis of Canine Procalcitonin Protein Polyclonal Antibody

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