稳定表达人乳头瘤病毒16E7蛋白HaCaT细胞株的建立

Establishment of a human keratinocyte cell line （HaCaT） stably expressing human papillomavirus type 16 E7 （HPV16E7） protein

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。将重组质粒转染人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297bp条带；Western印迹可检测到E7蛋白稳定表达。结论成功构建m稳定表达HPV16E7蛋白的HaCaT细胞株。

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 HPVI6E7 cDNA, and Western blot confirmed the stable expression of HPV16E7 protein. Conclusion A human keratinocytc cell line （HaCaT） stably expressing HPV16E7 protein is suecessfully established.