摘要
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cDNA,并将扩增的cDNA片段插入pcDNA3.1(+)真核表达质粒,构建重组质粒pcDNA-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入到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pcDNA-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。
Objective To construct a stable transfeetant of human cervical cancer cell line HeLa that can highly express human novel gene （PCIA1）. Methods RT-PCR technique was applied to amplify the eDNA of PCIA1 gene from total RNA isolated from HUVEC. After sequenced, the open reading frame （ORF） of PCIA1 eDNA was cloned into eukaryotie expression vector peDNA3.1 （＋） to form the recombinant plasmid named as peDNA3.1-PCIA1. Then lipofeetamine 2000 was used to transfeeted the peDNA3.1-PCIA1 into HeLa cells. The PCIA1-transfeeted 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 transfeetant of PCIA1.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第3期335-338,共4页
Journal of Sichuan University(Medical Sciences)
基金
*国家自然科学基金(批准号30130260
30300408)资助
