新基因PCIA1真核表达载体的构建及HeLa细胞稳定转染株的建立与鉴定

目的构建人的新基因（PCIA1）的真核表达载体，并将其载体稳定转染到HeLa细胞株中。方法以人脐静脉内皮细胞（HUVEC）总RNA为模板，通过RT—PCR扩增PCIA1基因编码区eDNA，并将扩增的eDNA片段插入peDNA3．1（＋）真核表达质粒，构建重组质粒peDNA—PCIA1，重组子经酶切分析及测序鉴定后，用脂质体转染技术将该表达载体导人到HeLa细胞中，经G418筛选并建立稳定的转染细胞株，用RT-PCR和Westernblot检测PCIA1基因在HeLa细胞中的表达。结果经RT—PCR及Westernblot检测，证实真核表达重组质粒peDNA-PCIA1构建成功，PCIA1基因已经稳定转染到了HeLa细胞中并表达PCIA1蛋白。结论成功建立了人新基因PCIA1的稳定转染细胞株，为进一步研究PCIA1的功能奠定了基础。

The Establishment and Identification of the Stable Transfectant of HeLa Cell Expressing Human New Gene( PCIA1)

Objective To construct a stable transfectant of human cervical cancer cell line HeLa that can highly express human novel gene (PCIA1). Methods RT-PCR technique was applied to amplify the cDNA of PCIA1 gene from total RNA isolated from HUVEC. After sequenced, the open reading frame (ORF) of PCIA1 cDNA was cloned into eukaryotic expression vector pcDNA3.1(+) to form the recombinant plasmid named as pcDNA3.1-PCIA1. Then lipofectamine 2000 was used to transfected the pcDNA3.1-PCIA1 into HeLa cells. The PCIA1-transfected HeLa cells were selected with G418, for 4 weeks. Finally, RT-PCR and Western blot were used to detect the expression of human PCIA1 in HeLa cells. Results By RT-PCR and Western blot, we found that the human novel gene PCIA1 stably expressed in HeLa cells. Conclusion This study indicated that human PCIA1 could be stably expressed in HeLa cells and its functions on tumor cells could be further analyzed by this stable transfectant of PCIA1.