表达人CD36基因的293T细胞株的建立

Establishment 293T cell lines expressing human CD36 gene

目的 建立一种表达人类CD36基因的293T细胞株,用于CD36抗原的研究及CD36抗体检测.方法 采用RT-PCR技术,从人血小板中获取CD36基因的cDNA,克隆至表达载体pEGFP-C1中,对重组质粒测序鉴定后将质粒转染至293T细胞,该转基因的产物在293T细胞中的表达被观察超过1年.与CD36单克隆抗体及CD36阳性血清反应的流式细胞术试验评估这个细胞株的敏感性和特异性.结果 重组表达载体pEGFP-C1-CD36被成功构建,流式细胞术验证其转染效率达90％以上,实时荧光定量PCR和Western blot证明CD36基因在293T细胞中过表达.流式细胞试验结果为CD36单克隆抗体及CD36阳性血清为阳性,而其他所有的血清样本,包括CD36抗体阴性血清、含有HLA或HPA-3a抗体血清均为阴性.结论 建立的pEGFP-C1-CD36-293T细胞株能成功高效表达CD36抗原,并能特异快速、成功地检测CD36抗体,并且不受其他血型抗体的干扰,有一定的运用前景.

Objective To establish cell lines expressing human CD36 gene.Methods cDNA of CD36 gene from human platelet was obtained by reverse transcription polymerase chain reaction （RT-PCR）.CD36 gene was cloned to eukaryotic expression vector pEGFP-C1.The cloning result was confirmed by sequencing.The recombinant plasmid was introduced into 293T cells.Flow cytometry was used to detect the transfection efficiency,and real-time quantitative PCR and western blot was used to determine the CD36 expression level.Expression of CD36 gene in 293T cells has been observed for more than one year.CD36 positive serum and CD36 monoelonal antibody were used to evaluate the sensitivity and specificity of cell lines by flow cytometry.Results The recombinant pEGFP-C1-CD36 vector was successfully constructed.The efficiency of transfection with pEGFP-C1-CD36 in 293T was up to 90%.Real-time quantitative PCR confirmed CD36 gene over-expression in 293T cells.Flow cytometry showed that positive reactions were obtained in the sample containing CD36 antibody serum and CD36 monoclonal antibody.All the other serum samples containing HLA or HPA-3a antibodies and CD36 negative serum showed negative result.Conclusion The established cell lines successfully express CD36,which has high specificity in the detection of CD36 antibody.