CAJ | 학술논문

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PeDNA3.1(+)Ⅲa和PeDNA3.1(+)ⅢanT1565C.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peDNA3.1(+)连接,构建突变型质粒PeDNA3.1(+)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PeDNA3.1Ⅲa重组质粒,转染至CHO经FCM检测均有GPⅢa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(+)Ⅲa T1565C;②获得野生型和突变型GPⅢa的CHO细胞系.
목적 구건인야생형화돌변형혈소판당단백Ⅲa(GPⅢa)진핵표체중조질립PeDNA3.1(+)Ⅲa화PeDNA3.1(+)ⅢanT1565C.방법 선택인홍백혈병세포제취인기인조총RNA,경과역전록-취합매련반응후,채용정점유변기술,통과삼륜취합매련식반응장제2외현자1565위기인유T치환위C,파최종산물여질립peDNA3.1(+)련접,구건돌변형질립PeDNA3.1(+)Ⅲa.결합동시구건적야생형PcDNA3.1(+)Ⅲa,채용양리자지질체법진행공전염도CHO세포중,통과류식세포술감정야생형화돌변형PcDNA3.1(+)Ⅲa재CHO세포적표체정황.결과 통과DNA측서,성공획득인야생형화돌변형PeDNA3.1Ⅲa중조질립,전염지CHO경FCM검측균유GPⅢa표체.결론 ①성공구건료야생형화돌변형진핵표체재체PcDNA3.1(+)Ⅲa화PcDNA3.1(+)Ⅲa T1565C;②획득야생형화돌변형GPⅢa적CHO세포계.
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.