CAJ | 학술논문

目的建立稳定表达人乳头瘤病毒(HPV)16E7蛋白的HaCaT细胞株.方法利用PCR及酶切技术扩增出CaSki细胞中HPV16E7基因并定向克隆到真核细胞表达载体pcDNA3.1(+)中,构建真核表达质粒pcDNA3.1(+)-HPV16E7.将重组质粒转染入HaCaT细胞,经G418筛选稳定表达HPV16E7蛋白的细胞株并予以鉴定.结果经酶切、测序鉴定构建pcDNA3.1(+)-HPV16E7成功.RT-PCR扩增产物经琼脂糖凝胶电泳后检测到HPV16E7mRNA表达的特异性297 bp条带;Western印迹可检测到E7蛋白稳定表达.结论成功构建出稳定表达HPV16E7蛋白的HaCaT细胞株.
목적건립은정표체인유두류병독(HPV)16E7단백적HaCaT세포주.방법이용PCR급매절기술확증출CaSki세포중HPV16E7기인병정향극륭도진핵세포표체재체pcDNA3.1(+)중,구건진핵표체질립pcDNA3.1(+)-HPV16E7.장중조질립전염입HaCaT세포,경G418사선은정표체HPV16E7단백적세포주병여이감정.결과경매절、측서감정구건pcDNA3.1(+)-HPV16E7성공.RT-PCR확증산물경경지당응효전영후검측도HPV16E7mRNA표체적특이성297 bp조대;Western인적가검측도E7단백은정표체.결론성공구건출은정표체HPV16E7단백적HaCaT세포주.
Objective To establish a human keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein. Methods HPV16E7 gene was amplified from CaSki cells using PCR and inserted into the eukaryotic expression plasmid pcDNA3.1. Then, the recombinant expression plasmid pcDNA3.1-HPV16E7 was transfected into HaCaT cells followed by G418 selection and identification by RT-PCR and Western blot. Results The recombinant eukaryotic expression plasmid pcDNA3.1-HPV16E7 was successfully identified by restriction enzyme digestion pattern and sequence analysis. Agarose gel electrophoresis of RT-PCR products detected the 297-bp fragment of HPV16E7 cDNA, and Western blot confirmed the stable expression of HPV16E7 protein. Conclusion A human keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein is successfully established.