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

摘要/Abstract

摘要： 试验旨在获得高纯度犬降钙素原（procalcitonin，PCT）重组蛋白，并对该蛋白的临床应用进行分析。采用大肠杆菌表达外源蛋白的方法，将PCT基因克隆至原核表达载体pET-30a（+）中，经双酶切和测序鉴定阳性的重组质粒转化大肠杆菌BL21（DE3）感受态细胞，以终浓度为1 mmol/L的IPTG诱导表达，采用镍柱进行重组蛋白的纯化，纯化蛋白免疫小鼠制备多克隆抗体，间接ELISA法检测抗体效价，Western blotting检测重组蛋白与犬PCT多克隆抗体的特异性，采用ELISA法检测健康犬和炎症犬血清PCT。双酶切及测序结果显示，犬PCT基因成功插入pET-30a（+），未出现碱基突变；SDS-PAGE结果显示，重组蛋白以可溶性形式表达，分子质量为14.9 ku，间接ELISA法测定的免疫小鼠血清抗体效价达1：51 200。Western blotting结果表明，制备的多抗血清具有很好的特异性；ELISA结果显示，炎症犬血清内检测到PCT，且其浓度与炎症反应程度呈正相关。结果表明，本研究成功制备了犬PCT蛋白多克隆抗体，且犬血清PCT蛋白是一种潜在的新型严重细菌性炎症感染指示物。

关键词: 降钙素原(PCT); 原核表达; 纯化; 多克隆抗体制备

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

中图分类号:

Q786

王栋, 刘营, 张康, 张鸿焱, 苏峰, 马卫明. 犬降钙素原蛋白多克隆抗体制备及应用分析[J]. 中国畜牧兽医, 2020, 47(3): 892-901.

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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