人乳头瘤病毒E7与人干扰素α-2b融合基因在中国仓鼠卵巢细胞中的表达

The construction of eukaryotic expression plasmid of HPV11-E7/human IFNα-2b fusion gene and its expression in CHO

目的:构建人乳头瘤病毒(HPV)11-E7及人干扰素(IFN)α-2b的真核表达载体,并在中国仓鼠卵巢(CHO)细胞中表达。方法:同时用KpnⅠ、EcoRⅠ酶切pET-32a-IFNα-2b-linker-HPV11-E7及pcDNA3.1,将酶切产物纯化回收,两者的回收产物进行连接反应。连接产物转化大肠杆菌感受态细胞DH5α,随机挑选阳性克隆并提取质粒酶切鉴定;用脂质体法转染CHO细胞,用免疫荧光法检测融合基因的表达。结果:DNA测序结果显示,成功地将IFN-α-2b-linker-HPV11-E7装入pcDNA3.1的KpnⅠ、EcoRⅠ酶切位点之间,且其序列与设计完全一致;激光扫描共聚焦显微镜观察可见目的蛋白的表达。结论:成功地构建了HPV11-E7及IFNα-2b的真核表达载体,并在CHO细胞中表达,为提高DNA疫苗免疫效果的研究奠定了基础。

Objective： To construct the expression plasmid of HPV11-ET/ human IFNα-2b fusion gene and express the fu- sion gene in Chinese Hamster Ovary （CHO） cell. Methods： pET-32α-IFNα-2b-linker-HPV11-E7 and pcDNA3.1 were digested by Kpn Ⅰ and EcoR Ⅰ, digested products were linked by T4 DNA ligase. DH5α was transformed with the linked product, positive clones were identified after Kpn Ⅰ and EcoR Ⅰ digestion. The recombinant plasmid carrying HPV11-E7/ human IFNα-2b fusion gene was introduced into CHO cells by lipofectin. The CHO cell containing pcDNA3.1 （＋）-IFNα-2b-linker- HPV11-E7 was examined by immunofluorescence staining. Results： The DNA sequencing showed that HPV11-ET/ human IFNα-2b fusion gene was successfully inserted into the pcDNA3.1（＋） plasmid between Kpn Ⅰ and EcoR Ⅰ. Conclusion： The successful construction of eukaryotic expression plasmid of HPVll-ET/ human IFNα-2b fusion gene and its expression in CHO cells may establish the base for increasing immunological result of DNA vaccine.