摘要
为研究融合表达肿瘤靶向肽(RGD)、细胞穿膜肽(TAT)和白树毒素(GEL)基因重组减毒沙门菌对乳腺癌细胞MDA-MB-231的体外抑制作用,将重组质粒载体pEGFP-RGD-TAT-GEL导入至减毒沙门菌LH430中,构建了重组减毒沙门菌LH430-RGD-TAT-GEL。经PCR、双酶切鉴定正确后,将重组菌转染MDA-MB-231细胞,提取总RNA后进行反转录,采用PCR技术检测目的基因的转录情况,细胞增殖毒性试验检测重组菌对MDA-MB-231细胞和HEK-293细胞的抑制率。结果显示:被重组菌转染后的MDA-MB-231细胞经RT-PCR检测,目的基因高效表达,对MDA-MB-231细胞最佳抑制条件为细菌浓度1×10^8 CFU/μL,转染时间为48 h,且半数抑制浓度为4.643×10^7CFU/μL。本试验为研究细胞穿膜肽、核糖体失活蛋白和细菌联合治疗肿瘤的后续动物试验提供试验支持。
In order to determine the in vitro inhibitory effect of recombinant attenuated Salmonella expressing tumor targeting peptide(RGD),cell penetrating peptide(TAT)and Bai Shu toxin(GEL)genes on breast cancer cell MDA-MB-231,the recombinant plasmid vector pEGFP-RGD-TAT-GEL was introduced into the attenuated Salmonella LH430 to construct recombinant attenuated Salmonella LH430-RGD-TAT-GEL.The recombinant bacteria were correctly identified by PCR and double enzyme digestion,and are then transfected into MDA-MB-231 cells.Next,the total RNA was extracted and reverse transcribed,the transcription of the target gene was detected using the PCR technology.Finally,the inhibition rate of the recombinant bacteria on MDA-MB-231 cells and HEK-293 cells was detected by cell proliferation toxicity test.The results showed that MDA-MB-231 cells transfected by the recombinant bacteria were detected by RT-PCR,and the target gene was highly expressed.The optimal inhibition conditions for MDA-MB-231 cells were a bacterial concentration of 1×10^8 CFU/μL,transfection time of 48 h,and a half inhibition rate of 4.643×10^7 CFU/μL.This experiment provided experimental support for the follow-up animal experiments on the combined treatment of tumor with cell penetrating peptide,ribosome inactivating protein and bacteria.
作者
蓝肇煜
王小月
李丹
武静桥
范真玮
姚景丽
赵微
何敬文
陈婷
金天明
LAN Zhaoyu;WANG Xiaoyue;LI Dan;WU Jingqiao;FAN Zhenwei;YAO Jingli;ZHAO Wei;HE Jingwen;CHEN Ting;JIN Tianming(College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300384,China)
出处
《畜牧与兽医》
北大核心
2020年第10期78-84,共7页
Animal Husbandry & Veterinary Medicine
基金
国家自然基金项目(31572492)
天津市科技计划项目(高端兽药先进制造科技重大专项)(17ZXGSNC00030)
天津市“兽医生物技术创新团队”资助项目(TD13-5091)。
