补体免疫抑制剂-免疫球蛋白超家族补体受体的原核表达与纯化  

The expression and purification of a new complement inhibitor complement receptor of the immunoglobulin superfamily

作　　者：陶思跃 尤涛 毕意辉 苏盖 Tao Siyue;You Tao;Bi Yihui;Su Gai(Department of Orthopedics,Anhui Provincial Hospital Affiliated to Anhui Medical University,Hefei 230036,China)

机构地区：[1]安徽医科大学附属省立医院骨外科,合肥230036

出　　处：《中华实验外科杂志》2019年第7期1267-1269,共3页Chinese Journal of Experimental Surgery

基　　金：安徽省重点研究与开发计划项目(201904a07020107).

摘　　要：目的构建免疫球蛋白超家族补体受体(CRIg)的原核表达质粒pSmartⅠ-SUMO-CRIg,并通过大肠杆菌表达体系对其进行表达和纯化,并检测表达产物活性.方法从人肝脏细胞HHMa中提取总RNA,采用反转录-聚合酶链反应(RT-PCR)和DNA无缝克隆技术,将编码人CRIg胞外段基因序列克隆至pSmart-Ⅰ载体上,选择阳性菌落进行质粒提取和测序,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Westernblot)对其表达产物进行检测,预测SDS-PAGE检测结果位于40 000处.最后通过补体旁路途径溶血实验检测其活性.重复3次实验取算术平均值绘制溶血曲线.结果扩增出的人CRIg片段长度为819 bp,测序结果比对为人CRIg胞外段序列,提取质粒转化至Rosetta菌株后通过IPTG诱导发现CRIg可以稳定在上清中表达.溶血实验证明25 μm蛋白浓度可以几乎完全抑制溶血.结论 CRIg可以在原核表达体系中实现可溶性表达.Objective To construct the prokaryotic expression plasmid pSmartⅠ-SUMO-complement receptor of the immunoglobulin superfamily (CRIg) of the CRIg,next express and purify it by E.coli expression system,finally detect the activity of the expressed product.Methods Total RNA was extracted from human liver cell HHMa,and the gene sequence encoding the extracellular domain of human CRIg was cloned onto the pSmart-Ⅰ vector by reverse transcriptase-polymerase chain reaction (RT-PCR) and Seamless cloning.Positive colonies were selected for plasmid extraction and sequencing,and the expressed products were detected by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting.Finally,the activity was detected by alternative pathway hemolysis assay.The experiment was repeated three times and averaged to plot the hemolysis curve.Results The PCR product amplified from human CRIg was 819 bp in length.The sequencing result was aligned with the extracellular segment of human CRIg.After the plasmid was transformed into Rosetta strain,it was found by IPTG that CRIg could be stably expressed in the supernatant.Hemolysis experiments demonstrated that a 25 μm protein concentration could almost completely inhibit hemolysis.Conclusion CRIg can achieve soluble expression in prokaryotic expression system.

关 键 词：免疫球蛋白超家族补体受体基因 补体 免疫抑制剂 无缝克隆 原核表达载体 

分 类 号：R392[医药卫生—免疫学]