重组穿梭质粒pIMP1-eHER2/neu的构建及其生孢梭菌稳定转化株的筛选与鉴定  

Construction of recombinant shuttle plasmid pIMP1-eHER2/neu and screening and identification of its stable Clostridium sporogenes transformants

作　　者：张艳丽[1] 张文卿[1] 王秋波[1] 丁守怡[1] 吕锐[1] 孟林[1] 

机构地区：[1]青岛大学医学院病原生物学实验室,山东青岛266071

出　　处：《细胞与分子免疫学杂志》2011年第11期1184-1187,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：山东省医药卫生科技发展计划项目(2009HD005)

摘　　要：目的:制备人原癌基因HER2/neu的细胞外区域(ECD)基因修饰的重组生孢梭菌,为进一步研究其抗肿瘤作用奠定基础。方法:利用SOE-PCR将HER2/neu ECD基因连接到厌氧菌的内β1,4-葡聚糖酶的启动子和信号肽基因(eg-lAp)下游,构建融合基因eglAp-HER2/neu;将其插入大肠杆菌-厌氧菌穿梭质粒pIMP1,构建重组质粒;首先将重组质粒导入大肠杆菌DH5α内并进行鉴定;然后用电穿孔法将重组质粒导入生孢梭菌。利用红霉素抗性筛选重组生孢梭菌;采用菌液PCR鉴定阳性克隆。结果:酶切和测序结果显示,插入质粒pIMP1内的eglAp-HER2/neu融合基因序列及读框正确。菌液PCR结果表明,重组质粒pIMP1-eHER2/neu成功转化生孢梭菌;经过20多代抗生素压力筛选,重组生孢梭菌仍能稳定携带pIMP1-eHER2/neu。结论:成功制备了重组质粒pIMP1-eHER2/neu生孢梭菌的稳定转化株,为其进一步的抗肿瘤作用研究奠定基础。AIM： To construct recombinant clostridium sporogenes modified with the extracellular domain of human oncogene HER2/neu, to lay a foundation for further study of its antitumor effect. METHODS： The extracellular domain （ECD） of HER2/neu gene was attached to the downstream of promoter and signal sequence of clostridia endo-1,4-glucanase （eglAp） by SOE-PCR to construct fusion gene eg- IAp-HER2/neu, which was then inserted into E. coli-clostridia shuttle plasmid pIMP1 to construct recombinant plasmid plMPl-eHER2/neu. The recombinant plasmid was firstly transformed into E. coil DHSα. Then the correct construct was identified and introduced into C. sporogenes by electroporation. Positive clones were selected by erythromycin re- sistance, bacteria PCR were used for verification. RESULTS： Restriction map and sequencing result showed that the sequence and ORF of fusion gene eglAp-HER2/neu in recombinant plasmid pIMP1-eHER2/neu was correct. Bacteria PCR results indicated that the recombinant plasmid pIMP1-eHER2/neu was successfully transformed into C. sporogenes. After more than 20 passages under antibiotic pressure, C. sporogenes transformants could stably carry the recombinant plasmid pIMP1-eHER2/neu. CONCLUSION： Stable C. sporogenes transformants with the recombinant plasmid plMPl-eHER2/neu are successfully acquired, which laid a foundation for further anti-tumor study.

关 键 词：大肠杆菌-厌氧菌穿梭质粒 生孢梭菌 HER2/NEU ECD pIMP1 

分 类 号：R392.12[医药卫生—免疫学]