Construction and identification of wild-type and mutational platelet glycoprotein M a in eukaryotic expression vector

Objective To construct the eukaryotic expression vectors of wild-type and mutational pcD-NA3.1 (+)Ⅲa. Methods RNA was extracted from the cell of human erythroleukemia, using the PCR site directed mutagenesis technique after reverse etranscriptase polymerase chain reaction (RT-PCR), the base gene 1565 T of the second exon was permuted to C through three cycles of polymerase chain reaction(PCR) ,and the ultimate amplified products were binded with the vector pcDNA3.1(+) to conduct mutational pcDNA3.1(+)Ⅲa. The wild-type and mutational recombinants of pcDNA3.1(+)Ⅲa were transfected into Chinese hamster o-vary (CHO) cells by Lipofectamine 2000. Then the CHO cell lines were examed by FCM to detect the expression of wild-type and motational pcDNA3.1(+)Ⅲa in vitro. Results By sequence analysis ,the wild-type and mutational pcDNA3.1(+)Ⅲa were constructed successfully and were detected in transfected CHO cells by FCM. Conclusions ① Succeeded in constructing the eukaryotic expression vector of wild-type and mutational pcDNA3. 1(+)Ⅲa.②Succeeded in getting the stable expression of CHO cell line of wild-type and motational GPⅢa. Key words: Polymerase chain reaction; Platelet membrane glycoprotein Ⅲa; pcDNA3.1(+)