HPV16 E7“自杀性”DNA疫苗的构建及其体外表达特性研究

Construction and Expression Characteristics in vitro of Human Papillomavirus Type 16 E7 Suicidal DNA Vaccine

目的构建人乳头瘤病毒16型(HPV16)E7基因的"自杀性"DNA疫苗,并研究其体外表达能力及诱导细胞凋亡作用。方法以pET32/E7质粒为模板,用PCR方法扩增HPV16 E7基因,构建真核表达重组质粒pSCA/E7,经PCR、酶切和测序鉴定正确后,将重组质粒转染BHK-21细胞,然后用免疫荧光技术检测HPV16 E7在细胞中的表达,用dUTP缺口末端标记技术(TUNEL)进行染色检测其诱导细胞凋亡作用。结果PCR扩增得到约400 bp的目的基因片段,测序结果与GenBank中的东亚型HPV16 E7基因序列100%同源。免疫荧光技术检测证实,pSCA/E7重组质粒能在BHK-21细胞中表达目的基因。TUNEL检测证实,pSCA/E7重组质粒能诱导被转染的BHK-21细胞发生凋亡。结论成功构建HPV16 E7基因的重组质粒pSCA/E7,该重组质粒可在BHK-21细胞中表达,并能诱导被转染的细胞发生凋亡。

Objective To construct the suicidal DNA vaccine of human papillomavirus type 16 E7 gene （HPV16）, and explore the DNA vaccine expression characteristics in vitro and capacity of inducing the transfected cells into apoptosis. Methods HPV16 E7 gene cloned by PCR from pET32/E7 was inserted into the plasmid pSCA1 to construct the recombinant plasmid pSCA/E7, followed by identification with PCR, BamH Ⅰ and Sma Ⅰ digestion and sequencing, pSCA/E7 was then used to transfect BHK-21 cell line. The transient expression of HPV16 E7 gene was confirmed by immuno-fluorescent staining, and the apoptosis induced by pSCA/E7 was checked with TDT-mediated dUTP nick end-labeling （TUNEL）. Results The cloned E7 gene fragment was about 400 bp in length. PCR, restriction endonuclease digestion and sequence analysis revealed that the HPV16 E7 gene was cloned into the eukaryotic expression plasmid pSCA1 successfully. Immunofluorescent staining confirmed that the E7 gene could express in BHK-21 cell line. The BHK-21 cells transfected with pSCA/E7 could be induced into apoptosis which was confirmed by TUNEL. Conclusion The results show that HPV16 E7 suicidal DNA vaccine can express in BHK-21 cell line, and induce the pSCA/E7 transfected cells into apoptosis. These findings may provide the foundation for exploring the therapeutic vaccine against HPV16-associated cervical cancer.