膜联蛋白A5真核表达质粒的构建

CONSTRUCTION OF RECOMBINANT PLASMID PEGFP-ANXA5

目的:构建在哺乳动物细胞中表达的膜联蛋白A5(anxA5)的重组质粒。方法:Trizol法从人胎盘中抽提细胞总RNA,RT—PCR法反转录为cDNA,以cDNA为模板,扩增膜联蛋白A5的基因序列,引入酶切位点．XhoI和BamHI。将真核表达载体pEGFP—N1和anxA 5的PCR产物分别双酶切后用T4连接酶连接,转化大肠杆菌后抽质粒,测序鉴定。结果:重组质粒测序后,与genebank中anxA5的mRNA进行比对,证明anxA5基因序列正确,质粒构建成功。结论:成功构建了pEGFP—anxA5重组质粒。为研究膜联蛋白A5的功能及其在细胞系中的作用奠定了基础。

Objective： To construct the recombinant plasmid pEGFP-anxA5 which is capable of expressing in mammalian cells for further study. Methods： Annexin AS gene containing XhoI and BamHI endoenzyme sites were obtained by using RT-PCR. Double enzyme digestion was conducted for green fluorescent protein vector pEGFP-N1 and RT-PCR product of annexin AS gene. Both fragments were connected by using T4 ligase and transferred to DH5α. The recombinant plasmids were abstracted and sequenced. Results： Identified by enzyme digestion, PCR and sequencing confirmed successful construction of the recombinant plasmid. Conclusions： The recombinant plasmid pEGFP-anxA5 was successfully constructed, as may facilitate the study of the function of annexin A5 in carcinoma cell lines.