重组大肠埃希菌-厌氧梭菌穿梭质粒pIMP1-eIL-12稳定转化生孢梭菌  

Stable transformation of E.coli-clostridia shuttle plasmid pIMP1-eIL-12 to clostridium sporogenes

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,青岛医学硕士266071

出　　处：《医学研究生学报》2012年第9期923-926,共4页Journal of Medical Postgraduates

基　　金：山东省医药卫生科技发展计划项目(2009HD005)

摘　　要：目的近些年来,以厌氧芽孢梭菌作为实体瘤基因治疗载体的研究逐渐受到关注。文中用重组人白细胞介素-12(rhIL-12)基因重组大肠埃希菌(E.coli)-厌氧梭菌穿梭质粒pIMP1,并稳定转化生孢梭菌,为增强杀伤肿瘤细胞的研究提供基础。方法用SOE-PCR法将rhIL-12基因连到厌氧梭菌的内源性β1,4-葡聚糖酶(endo-1,4-glucanase,eglA)启动子和信号肽之后,构建融合基因eglAp-rhIL-12,将其插入pIMP1,构建重组质粒pIMP1-eIL-12;重组质粒首先在E.coli DH5α内进行鉴定,然后用电穿孔法转化生孢梭菌;利用红霉素抗性筛选阳性克隆,并提取质粒鉴定阳性克隆。结果酶切和测序结果表明插入到pIMP1内的融合基因eglAp-rhIL-12序列及读框正确;抗生素压力筛选和20多代随机质粒提取酶切鉴定结果表明重组质粒pIMP1-eIL-12已稳定转化生孢梭菌。结论成功获得重组质粒pIMP1-eIL-12及其生孢梭菌稳定转化株,为以后的抗肿瘤研究奠定了基础。Objective In recent years, anaerobes of the genus clostridium has gradually gained attention as vectors in solid tumor gene therapy. This study aimed to recombine E. coliclostridia shuttle plasmid pIMP1 with the recombinant human IL12 （rhIL12） gene and stably transform clostridium sporogenes to enhance their antitumor effect. Methods The rhIL12 gene was attached to the promoter and signal sequence of clostridial endo1,4glucanase （eglAp） to construct the fusion gene eglAprhIL12, which was then in serted into pIMP1 to construct recombinant plasmid pIMPIeIL12. The construction was first tested in E. coli DHSo~, and then intro duced into C. sporogenes by electroporation. Positive clones were selected through erythromycin resistance, and plasmids extracted form the positive clones for verification. Results The restriction map and sequencing results showed that the sequence and ORF of the fu sion gene eglAprhIL12 inserted into pIMP1 was correct. Antibiotic pressure selection and restriction map of the plasmid randomly ex tracted from more than 20 passages confirmed that the recombinant plasmid pIMP1eIL12 had stably transformed C. sporogenes. Conclusion Recombinant plasmid plMPIelL12 and its stable C. sporogenes transformant were successfully obtained, which has paved the ground for further antitumor studies.

关 键 词：大肠埃希菌-厌氧梭菌穿梭质粒 生孢梭菌 rhIL-12 pIMP1 

分 类 号：R392.12[医药卫生—免疫学]