重组表达HIV不同亚型gp41蛋白的抗原表位暴露情况分析  

Evaluation of antigen epitope exposure status of recombinant HIV gp41 protein with different subtypes

作　　者：赵巧辉 李桂林 徐延伟 付光宇 吴学炜 杨增利 ZHAO Qiao-hui;LI Gui-lin;XU Yan-wei;FU Guang-yu;WU Xue-wei;YANG Zeng-li(Zhengzhou Immuno Biotech Co.,LTD.,Zhengzhou,Henan 450016,China;不详)

机构地区：[1]郑州伊美诺生物技术有限公司,河南郑州450016 [2]郑州安图生物工程股份有限公司,河南郑州450016

出　　处：《中国卫生检验杂志》2020年第1期15-17,30,共4页Chinese Journal of Health Laboratory Technology

摘　　要：目的通过化学发光法检测原核重组HIV I型4种不同亚型gp41膜外区蛋白对阴阳性标本的反应性,间接反映重组表达gp41膜外区蛋白的抗原表位暴露情况,为科研、临床异常标本分析提供分析方法。方法选取HIV I型4个国内主流基因型(BC、AE、B、C)膜外区序列,克隆至pGEX4T-3载体中,优化目标蛋白的纯化工艺,制备出HIV I型gp41膜外区相应蛋白作为包被抗原,与商品化gp41-HRP酶结合物配对后,夹心法进行HIV I型阴阳性标本反应性的测试,从而判断相应蛋白抗原表位暴露情况。结果HIV I型4个亚型的gp41膜外区相同区段在pGEX4T-3原核载体中均能表达出符合预期大小的目的蛋白;纯化后分别作为包被抗原,按相同浓度包被后进行HIV I型阴阳性标本的测试,结果显示不同亚型的gp41表达蛋白与商品化gp41-HRP酶反应性存在较大差异。结论通过原核表达系统高效表达了HIV I型主流亚型gp41膜外区蛋白,化学发光法组建夹心法进行阴阳性标本测试,其反应性不一致,间接反映出不同基因型选取同一表达区域进行体外重组表达其结构折叠方式有区别,此研究为HIV科研、临床标本验证等提供了基础。Objective Chemiluminiscence is used to detect the reactivity of 4 subtypes prokaryotic recombinant HIVI gp41 extracellular proteins to positive and negative samples.The results could indirectly reflect the antigen epitope exposure status and provide a method to analyze the abnormal samples on scientific and clinical research.Methods A total of 4 gp41 gene fragments of HIVI domestic mainstream genotypes(BC,AE,B,C)were selected and constructed to PGEX4T-3 vector by PCR and restriction enzyme digestion.The optimized purification method was used to obtain HIVI extramembrane gp41 proteins which worked as coating antigens.Then,the coating antigens and commercial gp41-HRP were used in the sandwich ELISA to estimate the reactivity of HIVI negative and positive samples.Thereby the exposure statuses of the corresponding protein epitopes were judged.Results The gp41 extramembrane proteins which were selected from the same extramembrane region in different genotypes could express target protein with expected mass weight via pGEX4T-3 prokaryotic vector.The purified proteins work as coating protein with the same concentration,proceed to test HIVI negative and positive samples,to evaluate the epitope exposure status of gp41 protein which was expressed by prokaryotic system.Conclusion The prokaryotic expression system efficiently expressed the gp41 outer membrane protein of HIV type I mainstream subtype.The chemiluminescence method was used to construct the sandwich method to test negative positive samples.The reactivity was inconsistent,which indirectly reflected that different genotypes selected the same expression region for recombinant expression in vitro.There are differences in structural folding methods,and this study provides the basis for HIV research and clinical specimen verification.

关 键 词：HIV I型 GP41 重组表达 膜外区蛋白 化学发光 

分 类 号：R446.6[医药卫生—诊断学]