HPV18E7重组质粒的构建及在大肠杆菌中的表达优化  

Construction of HPV18E7 recombinant plasmid and exploration of its optimization expression condition in Escherichia coli

作　　者：胡仁建[1] 蔡家利[1] 刘利[1] 涂漫语[1] 徐涛[1] 杜翠容[1] 罗佳[1] 丁森[2] 

机构地区：[1]重庆理工大学药学与生物工程学院,400054 [2]南京大学生命科学学院,210093

出　　处：《重庆医学》2013年第30期3647-3649,共3页Chongqing medicine

基　　金：重庆市教育委员会科技研究项目(KJ110820)

摘　　要：目的构建HPV18E7基因重组质粒,并探索其在大肠杆菌中的最佳表达条件。方法以提取的HeLa细胞株中的DNA为模板PCR扩增HPV18E7基因;将HPV18E7基因与载体pET-32a(+)连接为重组质粒pET-32a(+)-HPV18E7;将该重组质粒转入大肠杆菌BL21-DE3-pLysS细胞中,探索优化表达的条件,以获得大量HPV18E7致癌蛋白。结果 PCR扩增的目标基因大小序列与HeLa细胞中的HPV18E7基因的大小序列一致。用LB培养基,IPTG和乳糖诱导表达显示不同浓度、不同温度、不同诱导起始量等表达量均不高,尝试用ZYM-5052自动诱导培养基诱导,HPV18E7融合蛋白的表达量远远高于用异丙基-β-D-硫代吡唃半乳糖苷(IPTG)和乳糖诱导表达的表达量。结论测序正确的HPV18E7重组质粒在自动诱导培养基ZYM-5052中获得远远高于用异丙基-β-D-硫代吡唃半乳糖苷(IPTG)和乳糖诱导表达的HPV18E7融合蛋白。Objective To construct recombinant plasmids containing HPV18E7 gene ,and explore the optimization condition of its expression in Escherichia coli .Methods The genomic DNA extracted from HeLa cell line which served as a template to the HPV18 E7 gene was amplified using PCR method ;and the amplified product of HPV18E7 gene was connected to the pET-32a（＋ ） vector ,which composed the pET-32a（＋ ）-HPV18E7 recombinant plasmid ;the positive recombinant plasmids were transformed into BL21-DE3-pLysS competent cells and the optimized expression condition was explored in order to obtain a large amount of HPV18E7 oncogenic protein .Results The fragment length of PCR products of HeLa cell genomic DNA was consistent with that of HPV18 E7 gene .In LB medium ,the expression level of the target protein was not high under such conditions as different concentra-tion of IPTG and lactose ,different temperatures and different induction starting amount .Therefore the ZYM-5052 auto-induction medium was tried in this experiment ,and the expression amount of the fusion protein was much higher than that induced with IPTG and lactose .Conclusion The amount of HPV18E7 fusion protein in ZYM-5052 automatic induction medium is much higher than that induced with IPTG and lactose .

关 键 词：HPV18 HPV18E7基因 HELA细胞株 自诱导培养基 

分 类 号：R378[医药卫生—病原生物学]