重组cTnI-cTnC复合抗原的制备及其性能验证  

Preparation and performance validation of recombinant cTnI-cTnC complex antigen

作　　者：武强 史定刚 丁建静 WU Qiang;SHI Ding-gang;DING Jian-jing(Medicalsystem Biotechnology Co.,Ltd.,Ningbo 315104,Zhejiang Province,China)

机构地区：[1]美康生物科技股份有限公司,浙江宁波315104

出　　处：《中国生物制品学杂志》2020年第5期530-534,共5页Chinese Journal of Biologicals

基　　金：浙江省博士后择优资助项目(zj20180055)。

摘　　要：目的采用E.coli表达系统分别表达心肌肌钙蛋白I(cardiac troponin I,cTnI)和cTnC,制备cTnI-cTnC二元复合蛋白,并验证其抗原性能。方法将合成的cTnI和cTnC基因片段分别插入pET-22b和pET-28a原核表达载体,构建重组表达质粒,并分别转化E.coli BL21(DE3),IPTG诱导表达。将表达的cTnI和cTnC蛋白经Ni2+螯合亲和层析和离子交换层析两步纯化后,在Ca2+存在的条件下按摩尔比1︰1混合,制备cTnI-cTnC二元复合蛋白。采用双抗夹心ELISA法检测复合蛋白的抗原反应性;将cTnI单体和cTnI-cTnC复合蛋白稀释后,均分别于4和37℃条件下放置14 d,检测cTnI抗原放置前后的浓度变化,验证其储存稳定性。结果纯化的cTnI和cTnC重组蛋白的SDSPAGE纯度均达90%以上。与对照抗原(8IC63二元复合抗原)比较,cTnI-cTnC复合蛋白与抗体反应信号较强,且能够同时被cTnI和cTnC抗体识别;经37℃热加速试验后,cTnI单体蛋白浓度降低约80%,而cTnI-cTnC复合蛋白的浓度变化幅度在±15%间。结论制备的cTnI-cTnC二元复合蛋白具有良好的抗原反应性和储存稳定性,为下一步制备反应性良好和储存稳定的cTnI检测质控校准品提供了可靠的原料。Objective To express cardiac troponin I(cTnI)and cTnC in E.coli system,prepare cTnI-cTnC complex antigen and validate its performance.Methods The synthetic cTnI and cTnC genes were inserted into prokaryotic expression vectors pET-22b and pET-28a respectively,and the constructed recombinant plasmids were transformed to E.coli BL21(DE3)for induction with IPTG.The expressed cTnI and cTnC proteins were purified by nickel ion affinity chromatography and ion-exchange chromatography and mixed at a mole ratio of 1∶1 in presence of calcium ion.The antigenic reactivity of the prepared cTnI-cTnC complex protein was determined by double antibody sandwich ELISA.The cTnI and cTNI-CTnC complex protein were diluted and stored at 4 and 37℃for 14 d separately,of which the concentrations before and after storage were determined to validate the stability.Results Both the SDS-PAGE purity of purified cTnI and cTnC were more than 90%.Compared with control antigen(8IC63 complex antigen),CTnI-cTnC complex protein showed strong reaction signal with the antibody,and was recognized by cTnI and cTnC antibodies simultaneously.In accelerated stability test at 37℃,the concentration of cTnI protein decreased by about 80%,while the concentration change of cTnI-cTnC complex protein was±15%.Conclusion Prepared cTnI-cTnC complex protein showed good antigen reactivity and stability,which provided a reliable material for preparation of highly reactive and stable calibrators of cTnI assay kit.

关 键 词：心肌肌钙蛋白I 心肌肌钙蛋白C 大肠埃希菌 复合抗原 抗原反应性 储存稳定性 

分 类 号：Q819[生物学—生物工程]