【摘要】 目的:构建人雌激素受体β（hERβ）全长基因的真核表达载体pEGFP-C1-hERβ,并转染激素非依赖性前列腺癌细胞株PC-3M。方法:采用RT-PCR法从卵巢囊肿患者切除的部分正常卵巢组织中钓取hERβ全长基因（1 593 bp）,与pMD18-T载体连接做全自动序列测定。将测序正确的克隆质粒pMD18-T-hERβ和表达载体pEGFP-C1分别用HindⅢ和BamHⅠ进行双酶切,应用基因重组技术构建hERβ全长基因真核表达载体pEGFP-C1-hERβ。质粒经HindⅢ和BamHⅠ双酶切和PCR电泳鉴定后,采用脂质体转染法转染PC-3M细胞。结果:重组质粒经限制性酶切鉴定得到与hERβ全长基因长度一致（1 612 bp）的酶切产物;测序分析证实,PCR产物与GenBank上登录的hERβ基因（NM₀01437）序列完全一致,表明成功地完成了hERβ的扩增和表达载体的构建;荧光显微镜下可见转染的PC-3M细胞有绿色荧光蛋白的表达;PCR测定分析,转染细胞hERβ的表达增加。结论:构建完成真核表达载体pEGFP-C1-hER,βhERβ基因在PC-3M细胞内成功表达。更多还原

【Abstract】 Objective To construct an eukaryotic expression vector of human ERβ（hERβ） full length gene which named pEGFP-C1-hERβ and transfect it into hormone-independent prostate cancer cell line PC-3M.Methods The complete gene of hERβ（1 593 bp） was obtained with the technique of RT-PCR by using human ovary tissue mRNA as template which obstained from the ovarian cyst patients.The PCR product was connected with pMD18-T vector and sequenced automatically,and then the connected product and the expression vector pEGFP-C1 were digested by restrictive enzyme Hind Ⅲ and BamH Ⅰ,respectively,and the pEGFP-C1-hERβ vector was constructed by using gene recombinant technique.The plasmid was detected by endonuclease digestion and PCR,and then was transfected to PC-3M cells by lipid reagent.Results The clone of the complete hERβ gene and the construction of expression vector were finished successfully by endonuclease digestion and sequenced automatically.The product of the endomuclease digestion was as long as the human complete hERβ gene（1 612 bp）,and sequence analysis suggested that the hERβ sequence detected by PCR was identical to that published in GenBank（NM₀01437）.The expression of green fluorescence protein（GFP） was observed under the fluorescence microscope.Conclusion The eukaryotic expression vector pEGFP-C1-hERβ is constructed and it expresses in PC-3M cells successfully.更多还原