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

摘要

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. coli-clostridia shuttle plasmid pIMP1 with the recombinant human IL-12 ( rhIL-12 ) gene and stably transform clostridium sporogenes to enhance their antitumor effect. Methods The rhIL-12 gene was attached to the promoter and signal sequence of clostridial endo-1,4-glucanase ( eglAp ) to construct the fusion gene eglAp-rhIL-I2 , which was then inserted into pIMPI to construct recombinant plasmid pIMPI-eIL-12. The construction was first tested in E. coli DH5α, and then introduced 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 fusion gene eglAp-rhIL-12 inserted into pIMP1 was correct. Antibiotic pressure selection and restriction map of the plasmid randomly extracted from more than 20 passages confirmed that the recombinant plasmid pIMPl-eIL-12 had stably transformed C. sporogenes. Conclusion Recombinant plasmid pIMP1-eIL-12 and its stable C. sporogenes transformant were successfully obtained, which has paved the ground for further antitumor studies.%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.