野生型和突变型GPⅢa真核表达载体的构建与鉴定  

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

作　　者：张丽梅[1] 刘彦虹[1] 刘枫晨[1] 胡惠静[1] 

机构地区：[1]哈尔滨医科大学附属第二医院内分泌科,150086

出　　处：《国际免疫学杂志》2011年第4期321-324,共4页International Journal of Immunology

摘　　要：目的构建人野生型和突变型血小板糖蛋白Ⅲa（GPⅢa）真核表达重组质粒pcDNA3．1（＋）Ⅲa和pcDNA3．1（＋）Ⅲa^T1565C。方法选择人红白血病细胞提取人基因组总RNA，经过逆转录-聚合酶链反应后，采用定点诱变技术，通过三轮聚合酶链式反应将第2外显子1565位基因由T置换为C，把最终产物与质粒pcDNA3．1（＋）连接，构建突变型质粒pcDNA3．1（＋）Ⅲa。结合同时构建的野生型pcDNA3．1（＋）Ⅲa，采用阳离子脂质体法进行共转染到CHO细胞中，通过流式细胞术鉴定野生型和突变型pcDNA3．1（＋）Ⅲa在CHO细胞的表达情况。结果通过DNA测序，成功获得人野生型和突变型pcDNA3．1Ⅲa重组质粒，转染至CHO经FCM检测均有GP／Ⅲa表达。结论①成功构建了野生型和突变型真核表达载体pcDNA3．1（＋）Ⅲa和pcDNA3．1（＋）ⅢaT1565C；②获得野生型和突变型GPⅢa的CHO细胞系。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 ovary （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. （2）Succeeded in getting the stable expression of CHO cell line of wild-type and motational GPⅢa.

关 键 词：聚合酶链式反应 GPⅢa PCDNA3.1(+) 

分 类 号：R[医药卫生]