J4 ›› 2011, Vol. 10 ›› Issue (6): 21-24.
目的：为研究细胞周期蛋白在肿瘤形成过程的分子机制，构建带FLAG标签的细胞周期蛋白E的真核表达载体，并检
测其在瞬时转染HeLa细胞株中的蛋白表达。方法：通过RT—PCR扩增cyclinE基因编码cDNA，并将扩增的cDNA片段插入p3XFLAG—CMVTM-14 真核表达载体，重组子经酶切分析和测序鉴定后，用脂质体介导的基因瞬时转染法，将重组正确的表达载体转染HeLa细胞，用Western—blot技术检测其在HeLa细胞中融合蛋白的表达。结果：经酶切鉴定和测序分析证实人cvclin E的真核表达载体构建成功，并能在瞬时转染的HeLa细胞中表达。结论：成功构建了人cyclin E的真核表达载体，为进一步研究细胞周期蛋白的功能奠定了基础。
Objective：To construct eukaryotic expression vector with FLAG epitope highly expressing human cyclin E gene．Methods：Amplify the cDNA of cyclin E gene from total RNA isolated from HeLa cells using RT-PCR．After sequenced，the open reading frame(ORF)of cyclin E cDNA was cloned into eukaryotic expression vector p3XFLAG—CMVTM-14 to form a recombinant plasmid naIned as p3XFLAG- cyclin E．Then lipofectamine 2000 was used to transfected eukaryotic expression vectors into HeLa cells．Finally，Western—blot was applied to detect the expression of human cyclin E in HeLa cells．Results：Restriction enzyme digestion and nucleotide sequencing results confirmed that the recombinant plasmid was successfully constructed．By W estern-blot，we found that the human cyclin E were expressed in HeLa cells．Conclusion： W e cloned human cyclin E and constructed eukaryotic expression vector，which enable US do further research with cyclin proteins．
摘要：
目的：为研究细胞周期蛋白在肿瘤形成过程的分子机制，构建带FLAG标签的细胞周期蛋白E的真核表达载体，并检
测其在瞬时转染HeLa细胞株中的蛋白表达。方法：通过RT—PCR扩增cyclinE基因编码cDNA，并将扩增的cDNA片段插入p3XFLAG—CMVTM-14 真核表达载体，重组子经酶切分析和测序鉴定后，用脂质体介导的基因瞬时转染法，将重组正确的表达载体转染HeLa细胞，用Western—blot技术检测其在HeLa细胞中融合蛋白的表达。结果：经酶切鉴定和测序分析证实人cvclin E的真核表达载体构建成功，并能在瞬时转染的HeLa细胞中表达。结论：成功构建了人cyclin E的真核表达载体，为进一步研究细胞周期蛋白的功能奠定了基础。
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