人乳头瘤病毒6型全基因转染hTERT细胞  

Transfection of HPV6 Genome DNA into Human Keratinocyte Cell Line hTERT

作　　者：郭宗科[1] 王飞[2] 张红叶[2] 潘永正[2] 董正邦[2] 陈梅[2] 单莹[2] 严翘[2] 余卫平[3] 

机构地区：[1]东南大学附属中大医院整形科,江苏南京210009 [2]东南大学附属中大医院皮肤科,江苏南京210009 [3]东南大学医学院病理生哩学系,江苏南京210009

出　　处：《中国皮肤性病学杂志》2015年第5期443-446,共4页The Chinese Journal of Dermatovenereology

基　　金：江苏省自然科学基金(BK2012748);教育部留学回国人员科研启动基金(20121707)

摘　　要：目的探讨共转染与筛选技术获得稳定携带有HPV6全基因细胞的可能性,为HPV6全基因体外培养模型的建立奠定基础。方法质粒p EGFP-1用限制性内切酶切下EGFP段,凝胶电泳,纯化取掉EGFP段的DNA片段,T4 DNA连接酶进行线状DNA的自身环化,转染DH5a细胞,抗生素筛选,获去掉EGFP片段的新质粒p EGFP-△EGFP,多组内切酶酶切鉴定。限制性内切酶切下质粒p SP65-HPV6的HPV6全长基因,纯化后获HPV6全长线性基因。将线性HPV6基因和质粒p EGFP-△EGFP电转法共转染h TERT细胞,抗生素筛选、传代,Southern blot方法检测细胞内HPV6 DNA。结果HPV6基因组DNA转至h TERT细胞,经G418筛选1d,培养皿内可见对G418抗性的阳性细胞克隆出现,其形态与普通h TERT细胞相似。用Southern blot检测细胞内HPV11基因组的存在,传3代后,细胞内依然检测到HPV6 DNA存在。结论 HPV6基因组DNA可通过电转法成功导入h TERT细胞内,通过筛选可获得阳性细胞,HPV6 DNA在细胞内可保持3代以上。Objective To investigate the possibility that keratinocytes can stably maintain carrying of HPV6 genome by cotransfection and screening technology, for the basis of the establishment of the model of HPV6 gene in vitro. Methods Plasmid pEGFP-1 was digested with restricted enzyme and purified by gel electrophore- sis, and then the DNA fragment self-circulated with T4 DNA ligase. The religated DNA was transfected into coli DHSot and selected. With culture and analysis the positive clone, the plasmid pEGFP-AEGFP without the EGFP fragment was constructed. Plasmid pSP65-HPV6 was digested with EcoR I enzyme and release the HPV6 genome. The linear HPV6 was cotransfered with plasmid pEGFP-A EGFP into hTTERT cell with electroporation. Upon completion of the selection with G418, the Southern blot hybridization was applied to detect HPV6 DNA in the cotransfected cells （HPV6. hTERT cell）. Results The linear HPV6 was suc- cessfully induced into hTERT cell by eotransfeetion. HPV6 DNA was found in HPV6. hTERT cells with the Southern blot hybridization. Conclusion Our data indicate that HPV6 genome can be successfully intro- duced into hTERT ceils by transfection and HPV6 DNA-positive cells can be obtained by G418 selection. Moreover, HPV6 DNA is still present in the third passage of transfected ceils.

关 键 词：人乳头瘤病毒6 基因 转染 

分 类 号：R752[医药卫生—皮肤病学与性病学]