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

作　　者：相文忠[1] 王飞[2] 王群[3] 刘丰[4] 张兆松[4] 苏川[4] 毕志刚[1] 

机构地区：[1]南京医科大学第一附属医院皮肤性病科,江苏南京210029 [2]东南大学附属中大医院皮肤科,江苏南京210009 [3]广东省人民医院皮肤科,广东广州510080 [4]南京医科大学江苏省现代病原生物学重点实验室,江苏南京210009

出　　处：《临床皮肤科杂志》2006年第7期430-432,共3页Journal of Clinical Dermatology

基　　金：江苏省重点学科基金资助项目(135-03);国家自然科学基金资助项目(30100156)

摘　　要：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.

关 键 词：人乳头瘤病毒 E7基因 干扰素Α-2B 融合基因 

分 类 号：Q782[生物学—分子生物学] Q786