人血小板CD36转染细胞的建立及在CD36抗体检测中的应用  

Establishment of a cell line expressing the CD36 on human platelets and its application to the detec-tion of anti-CD36 antibodies

作　　者：徐秀章[1] Sentot Santoso 夏文杰[1] 丁浩强[1] 陈大伟[1] 邓晶[1] 陈扬凯[1] 王嘉励[1] 邵媛[1] 刘静[1] 叶欣[1] 

机构地区：[1]广州血液中心临床输血研究所,510095 [2]35385德国吉森大学临床免疫和输血医学研究所

出　　处：《中华微生物学和免疫学杂志》2016年第6期458-462,共5页Chinese Journal of Microbiology and Immunology

基　　金：广州市医药卫生科技项目（20141A011063）;广东省自然科学基金项目（2016A030313124,2016A030313123）;广州市科技计划项目（201607010007,201509010009）

摘　　要：目的：通过TA克隆和细胞转染技术建立稳定表达人CD36的转染细胞,并应用于CD36抗体检测。方法提取人血小板总RNA,逆转录为cDNA,经PCR扩增获得人血小板的CD36基因片段；TA克隆后转化TOP10 E. coli,蓝白斑筛选获得阳性重组子pcDNA3.1/V5-CD36,提取质粒DNA进行序列测定；将序列一致的质粒DNA在Effectene（Invitrogen）作用下转染HEK293T细胞；通过流式细胞仪分选建立稳定表达人血小板CD36的转染细胞,并利用该转染细胞对9份CD36抗体阳性标本进行流式和抗体捕获法（ACA）检测。结果获得稳定表达人血小板CD36的HEK293T转染细胞；与单克隆抗体捕获血小板抗原技术（ MAIPA）相比,利用CD36转染细胞建立的流式和抗体捕获法（ ACA）均可检出9份CD36抗体阳性标本,且操作简便。结论稳定表达人血小板CD36转染细胞的建立,为临床上CD36抗体的检测提供了有利工具,也为今后CD36在其他疾病中作用机制的研究提供实验资料。Objective To establish a cell line stably expressing the human CD36 by using TA clo-ning and cell transfection technology and to analyze its application to the detection of anti-CD36 antibodies. Methods Total RNA was isolated from human platelets and then used to synthesize complementary DNA （ cDNA） . Sequence of the gene encoding CD36 on human platelets was obtained by PCR amplification. The recombinant vector was transformed into TOP10 E. coli after TA cloning. The positive recombinant pcDNA3. 1/V5-CD36 plasmid was screened out by blue-white selection and then sequenced. The correctly constructed plasmid coated with Effectene？ Transfection Reagent was transferred into HEK293T cells. Fluo-rescence-activated cell sorting was performed to screen out the cell line that could stably express the CD36 on human platelets. The transfected cell line-based flow cytometry analysis and antibody capture assay （ ACA） were established and used for antibody detection in nine serum samples positive for anti-CD36 antibodies. Results The HEK293T cell line stably expressing the recombinant CD36 was successfully established. Compare with the monoclonal antibody immobilization of platelet antigens assay （ MAIPA） , anti-CD36 anti-bodies could be easily identified in nine serum samples by using the transfected cell line-based flow cytome-try analysis and ACA. Conclusion This study suggests that the HEK293T cells stably expressing the re-combinant CD36 could be used in flow cytometry analysis and ACA for the detection of anti-CD36 antibod-ies. It also paves the way for further researches on the mechanism of CD36 in other diseases.

关 键 词：CD36 HEK293T细胞 TA克隆技术 

分 类 号：R440[医药卫生—诊断学]