›› 2010, Vol. 30 ›› Issue (7): 797-.

• 论著(基础研究) • 上一篇    下一篇

人重组Oct3/4真核表达载体构建及在卵巢癌细胞中的表达研究

王 育, 肖世金, 赵爱民   

  1. 上海交通大学 |医学院仁济医院妇产科, 上海 200127
  • 出版日期:2010-07-25 发布日期:2010-07-26
  • 通讯作者: 赵爱民, 电子信箱: zam0526@yahoo.com.cn。
  • 作者简介:王 育(1970—), 女, 副主任医师, 博士;电子信箱: drwangyu@hotmail.com。

Construction of human recombinant Oct3/4 eukaryotic expression vectors and their expression in ovarian cancer cells

WANG Yu, XIAO Shi-jin, ZHAO Ai-min   

  1. Department of Obstetrics and Gynaecology, |Renji Hospital, |School of Medicine, Shanghai Jiaotong University, |Shanghai 200127, China
  • Online:2010-07-25 Published:2010-07-26

摘要:

目的 构建人Oct3/4真核表达载体并转染人卵巢癌细胞株(SKOV3),观察Oct3/4在SKOV3稳定细胞株中的表达。方法 采用RT-PCR技术扩增获得目的基因Oct3/4 cDNA产物,利用pcDNA3.1载体质粒构建人重组Oct3/4真核表达载体pcDNA3.1-Oct3/4,酶切及DNA测序鉴定。利用脂质体Lipofectamine 2000介导重组质粒转染SKOV3,以空载体转染细胞和未转染细胞作为转染对照组和阴性对照组。经G418(neomycin)筛选获得SKOV3稳定转染细胞株,Western blotting分析检测细胞Oct3/4蛋白的相对表达水平。结果 PCR扩增产物经12 g/L琼脂糖凝胶电泳鉴定目的基因条带大小约1 000 bp,与理论预期值一致。构建完成人重组 Oct3/4真核表达载体pcDNA3.1-Oct3/4,DNA测序结果与GenBank数据库进行比对序列一致。Western blotting检测显示,转染pcDNA3.1-Oct3/4组稳定转染细胞中Oct3/4蛋白相对表达量(10.225±0.987)显著高于转染对照组(1.713±0.896)和阴性对照组(校正值为1)(P<0.01)。结论 成功构建人重组Oct3/4真核表达载体并转染SKOV3,稳定转染细胞株中Oct3/4蛋白呈稳定过量表达。实验为进一步研究Oct3/4基因在卵巢癌发生、发展及耐药机制中的作用奠定了基础。

关键词: Oct3/4基因, 载体, 蛋白表达, 卵巢癌

Abstract:

Objective To construct human Oct3/4 eukaryotic expression vectors and transfect human ovarian cancer cell line SKOV3, and observe the expression of Oct3/4 in SKOV3 cells. Methods Destination gene (Oct3/4) cDNA products were obtained by amplification with RT-PCR. Human recombinant Oct3/4 eukaryotic expression vectors pcDNA3.1-Oct3/4 were constructed by pcDNA3.1 vector plasmid, and were identified by restriction enzyme digestion and DNA sequence analysis. Recombinant plasmid was transfected into SKOV3 cells with Lipofectamine 2000, and SKOV3 cells transfected with empty vectors and those without transfection were served as transfection control group and negative control group, respectively. Stable transfection cells were screened by G418 (neomycin), and Western blotting was employed to detect the relative expression of Oct3/4 protein in cells. Results Destination gene amplified by PCR was about 1 000 bp in length analysed by 12 g/L agarose gel electrophoresis, which met the expectancy. Human recombinant Oct3/4 eukaryotic expression plasmid vectors pcDNA3.1-Oct3/4 were constructed, and the result of DNA sequencing was consistent with that of GenBank. Western blotting analysis revealed that the relative expression of Oct3/4 protein in stable pcDNA3.1-Oct3/4 transfected cells (10.225±0.987) was significantly higher than that in transfection control group (1.713±0.896) and negative control group (1 for calibration value) (P<0.01). Conclusion Human recombinant Oct3/4 eukaryotic expression vectors were successfully constructed, SKOV3 was transfected, and Oct3/4 protein is stably overexpressed in stable transfected cells, which lays a foundation for the further study of roles of Oct3/4 gene in ovarian cancer development and drug-resistance to chemotherapy.

Key words: Oct3/4 gene, vector, protein expression, ovarian cancer