血小板血型抗原HPA-2在CHO细胞中的表达

Expression of human platelet antigens HPA-2a and 2b in CHO cells

目的 在 CHO细胞中表达血小板血型抗原 HPA- 2 a和 2 b。方法 从人巨核细胞系 MO7e中采用 RT- PCR方法扩增出血小板血型抗原 HPA- 2抗原位点所在的血小板糖蛋白 b氨基端 30 2个氨基酸的 c DNA,并将第 4 34位的 C突变为T,使得相应的氨基酸苏氨酸 (Thr)突变为蛋氨酸 (Met)。将 HPA- 2 a和 2 b基因插入真核表达质粒 pc DNA3.1(+) ,将重组质粒 p HPA2 a和 p HPA2 b以脂质体介导的转染方法导入 CHO细胞中 ,以 G4 18筛选获得抗性克隆 ,表达产物用瑞斯托菌素诱导血小板凝集试验的抑制作用测定活性。结果 获得了人血小板血型抗原 HPA- 2 a和 HPA- 2 b的基因 ,并在 CHO细胞中进行了表达。表达产物具有 v WF结合功能。结论 成功地表达了人血小板血型抗原 HPA- 2 a和 HPA- 2 b,对进一步研究血型抗原特定表位及功能。

Objective To express human platelet antigens HPA 2a and 2b in CHO cells.Methods\ The cDNA fragment coding for the residues 1 302 of HPA 2a was obtained by RT PCR from human megakaryocyte cell line MO7e.A Threonine for methionine 145 replacement of HPA 2a was done by mutagenesis using the overlap extension method,and the mutation was verified by sequencing.Recombinant expression vectors containing cDNA of HPA 2a and 2b were transfected into CHO cells by lipofectamin.The transformants were selected by G418.The activity of the recombinant protein was assayed by inhibition of ristocetin induced platelet aggregation method.Results\ cDNA of HPA 2a and 2b were obtained,and expressed in CHO cells.The protein had the vWF binding activity.Conclusion\ CHO cell lines steadily expressing HPA 2a and 2b are obtained,and the expressed proteins have vWF binding activity.It's useful for the detection of HPA 2 antibodies and the investigation of the mechanisms of ITP.