Establishment of a cell line expressing the CD36 on human platelets and its application to the detec-tion of anti-CD36 antibodies
10.3760/cma.j.issn.0254-5101.2016.06.011
- VernacularTitle:人血小板CD36转染细胞的建立及在CD36抗体检测中的应用
- Author:
Xiuzhang XU
;
Santoso SENTOT
;
Wenjie XIA
;
Haoqiang DING
;
Dawei CHEN
;
Jing DENG
;
Yangkai CHEN
;
Jiali WANG
;
Yuan SHAO
;
Jing LIU
;
Xin YE
- Publication Type:Journal Article
- Keywords:
CD36;
HEK293T cell;
TA cloning
- From:
Chinese Journal of Microbiology and Immunology
2016;36(6):458-462
- CountryChina
- Language:Chinese
-
Abstract:
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.
