CAJ | 학술논문

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。DNA片段插入p3XFLAG—CMV^TM-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。vclin 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cyclinE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。
목적：위연구세포주기단백재종류형성과정적분자궤제，구건대FLAG표첨적세포주기단백E적진핵표체재체，병검측기재순시전염HeLa세포주중적단백표체。방법：통과RT—PCR확증cyclinE기인편마cDNA，병장확증적。DNA편단삽입p3XFLAG—CMV^TM-14진핵표체재체，중조자경매절분석화측서감정후，용지질체개도적기인순시전염법，장중조정학적표체재체전염HeLa세포，용Western—blot기술검측기재HeLa세포중융합단백적표체。결과：경매절감정화측서분석증실인。vclin E적진핵표체재체구건성공，병능재순시전염적HeLa세포중표체。결론：성공구건료인cyclinE적진핵표체재체，위진일보연구세포주기단백적공능전정료기출。
Objective： To construct eukalyotic expression vector with FLAG epitope highly expressing human cyclin E gene. Methods： Amplify the eDNA of eye/in E gene from total RNA isolated from HeLa ceils using RT-PCR. After sequenced, the open reading frame （ORF） of cyclin E eDNA was cloned into eukaryotic expression vector p3XFLAG-CMV^TM-14 to form a recombinant plasmid named 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 cyelin E in HeLa cells. Results： Restriction enzyme digestion and nucleotide sequencing results confirmed that the recombinant plasmid was successfully construe, ted. By Western-blot, we found that the human eyclin E were expressed in HeLa cells. Conclusion： We cloned human cyclin E and constructed eukaryotic expression vector, which enable us do further research with cyclin proteins.