肿瘤抑制基因DOC2真核表达载体构建及在卵巢癌细胞HO8910中.docVIP

肿瘤抑制基因DOC2真核表达载体构建及在卵巢癌细胞HO8910中 　 作者：刘淑娟，韩军涛，辛晓燕，吴元明，陈苏民 【摘要】 　　目的 构建肿瘤抑制基因DOC2的真核表达载体pCDNA3.1p93，将其转入人卵巢癌细胞系HO8910中，对其基因表达进行相关检测，为进一步研究DOC2的功能奠定基础。方法 利用XhoI酶切含有p93cDNA的质粒得到p93cDNA 基因片段，将其连接入真核表达载体pCDNA3.1，并通过酶切鉴定。用脂质体介导法将真核表达载体pCDNA3.1p93 转染HO8910，通过G418筛选稳定表达克隆；用免疫组化法观察人DOC2蛋白在HO8910中的表达。结果 构建了DOC2的真核表达载体pCDNA3.1p93，并通过酶切鉴定。免疫细胞化学结果显示pIRES2EGFPp93成功转入HO8910中。结论 成功构建了真核表达载体pCDNA3.1p93并在HO8910中得到稳定表达，为研究DOC2蛋白的功能奠定了基础。 【关键词】 DOC2　真核表达载体　卵巢癌 　　Construction of The Eukaryotic Expression Vector pCDNA3.1p93 and Its Expression in Ovarian Cancer Cell Line HO8910 　　Abstract：Objective To construct an recombinant vector consisted of pCDNA3.1 with DOC2 cDNA(p93) and transfect it into human ovarian cancer cell line HO8910 in order to investigate the function of DOC2 gene on ovarian cancer cell.Methods The recombinant vector (pCDNA3.1p93) was constructed by XhoI and its correction was confirmed by restriction pattern. Then pCDNA3.1p93 was transfected into HO8910 by Lipofectamin and the positive clone 8910p93 and 8910pCDNA3.1 were selected by G418. Finally, the expression of DOC2 in 8910p93 was proved by immuncytochemical method.Results The recombinant vector pCDNA3.1p93 was constructed successfully. After being transfected into HO8910, the pCDNA3.1p93 positive clone could be harvested by G418 and the steady expression of protein DOC2 was confirmed by immuncytochemical method. Conclusion After the recombinant vector pCDNA3.1p93 being constructed, DOC2 gene could express steadily in HO8910.This will allow us to study the function of DOC2 gene in future. 　　Key words：DOC2; Eukaryotic expression vector; Ovarian cancer 　　0 引言 　　卵巢癌是妇科肿瘤中最常见的恶性肿瘤，其发病机制与抑癌基因的关系一直是该领域研究的热点[1]。1994年Mok等[2]在研究人正常卵巢上皮细胞与卵巢癌细胞的基因差异表达时，发现该基因在正常卵巢上皮细胞中有表达，而在卵巢癌细胞系及组织中无表达，故将其命名为DOC2（Differentially expressed in ovarian cancer 2）。人类DOC2位于染色体5p13，长约35kb，含有15个外显子和14个内含子，cDNA共3268bp，编码长度为770个氨基酸的蛋白质，分子量约为82.5KDa [3]。DOC2是具有信号转导功能的新的磷蛋白，它能抑制多种肿瘤细胞的生长，被认为是一种可能的肿瘤抑制基因，但其作