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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。
Objective To construct Clostridium sporogenes engineered bacteria with human IL-12 (hIL-12) gene and breast cancer target gene HER2 / neu extracellular domain (ECD) respectively, and to explore their roles in gene therapy of breast cancer. Methods The genes of hIL-12 and HER2 / neu ECD were ligated to the endogenous β-1,4-glucanase promoter and signal peptide sequence (eglAp) of Clostridium difficile to obtain the fusion gene eglAp-hIL -12 and eglAp-HER2 / neu. The fusion gene was inserted into E. coli-anaerobic shuttle plasmid pIMP1 to obtain recombinant plasmids pIMP1-ehIL-12 and pIMP1-eHER2 / neu. The recombinant plasmid was first transformed into E. coli DH5α, identified by restriction enzyme digestion and sequencing, and then electroporated into Clostridium sporosphaeroides; positive clones were screened by erythromycin resistance, and the positive clones were identified by bacterial liquid PCR and plasmid extraction, The target product in Clostridium sporogenes culture supernatant was analyzed by ELISA. Results The results of enzyme digestion and sequencing showed that the sequences of eukaryotic expression vectors such as eglAp-hIL-12 and eglAp-HER2 / neu and the reading frame of the recombinant plasmid were correct. Bacterial PCR, plasmid extraction and ELISA showed that the hIL-12 and HER2 / neu ECD genes were successfully Clostridium sporogenes modified and expressed. After more than 20 generations of antibiotic stress screening, Clostridium sporogenes can still stably carry and express foreign genes. Conclusion Clostridium sp. Clostridium strain modified by hIL-12 and HER2 / neu ECD genes was successfully obtained, which laid the foundation for further antitumor research.