hIL-12及HER2/neu ECD修饰的生孢梭菌工程菌的构建及鉴定  

Construction and identification of engineering Clostridium sporogenes modified with hIL-12 and HER2/neu ECD

作　　者：张艳丽[1] 张文卿[1] 王秋波[1] 丁守怡[1] 吕锐[1] 董海[2] 

机构地区：[1]青岛大学医学院病原生物学实验室,266071 [2]青岛大学医学院附属医院重症医学科,266000

出　　处：《免疫学杂志》2011年第11期987-991,共5页Immunological Journal

基　　金：山东省医药卫生科技发展计划项目(2009HD005)

摘　　要：目的构建人IL-12(hIL-12)基因和乳腺癌靶基因HER2/neu胞外区(ECD)分别修饰的生孢梭菌工程菌,探讨其在乳腺癌基因治疗中的作用。方法将hIL-12和HER2/neu ECD的基因分别连接到厌氧梭菌的内源性β-1,4-葡聚糖酶启动子和信号肽序列(eglAp)之后,得到融合基因eglAp-hIL-12和eglAp-HER2/neu。将融合基因插入大肠杆菌-厌氧菌穿梭质粒pIMP1,得到重组质粒pIMP1-ehIL-12和pIMP1-eHER2/neu。将重组质粒首先转化大肠杆菌DH5α,通过酶切和测序鉴定无误后,再用电穿孔法转化生孢梭菌;利用红霉素抗性筛选阳性克隆,采用菌液PCR和质粒提取鉴定阳性克隆,采用ELISA法对生孢梭菌培养上清中目的产物进行分析。结果酶切和测序结果表明重组质粒内的融合基因eglAp-hIL-12和eglAp-HER2/neu序列和读框正确,菌液PCR、质粒提取和ELISA结果显示hIL-12和HER2/neu ECD基因成功修饰生孢梭菌并表达。经过20多代抗生素压力筛选,重组生孢梭菌仍能稳定携带和表达外源基因。结论成功获得hIL-12和HER2/neu ECD基因分别修饰的生孢梭菌工程菌株,为进一步的抗肿瘤研究奠定基础。In the last few years,attention has been paid to using anaerobes of the genus clostridium as vectors for tumor gene therapy.For the sake of sending the gene of anti-tumor cytokine IL-12 and breast cancer target gene HER2/neu extracellular domain（ECD） to the center of tumor to exert a synergetic anti-tumor effect,the human IL-12（hIL-12） and HER2/neu ECD genes were inserted into E.coli-clostridia shuttle plasmid pIMP1 under the control of the promoter and signal peptide sequence of clostridium endo-β-1,4-glucanase gene（eglAp） to construct recombinant plasmids pIMP1-eIL-12 and pIMP1-eHER2/neu.The recombinant plasmids were firstly introduced into E.coli DH5α.Then the correct constructs were identified by enzyme digestion and sequencing and introduced into C.sporogenes by electroporation.Positive clones were selected by erythromycin resistance.Bacteria PCR and plasmid extraction were used for verification,and ELISA were used to detect the expressed products.The results showed that the sequence and ORF of fusion genes eglAp-hIL-12 and eglA-HER2/neu in recombinant plasmids are correct.hIL-12 and HER2/neu ECD genes were successfully modified C.sporogenes and expressed in it.After more than 20 passages under antibiotic pressure,C.sporogenes transformants could still stably carry and express the target gene.All these results indicated that stable C.sporogenes transformants modified with hIL-12 and HER2/neu ECD gene were successfully acquired,which would lay a foundation for further anti-tumor study.

关 键 词：hIL-12 HER2/NEU ECD pIMP1 生孢梭菌 

分 类 号：R737.9[医药卫生—肿瘤]