摘要
目的制备含pCMV-IL-2-IRES-NK4质粒减毒沙门菌TPIN并检测其稳定性。方法将pCMV-IL-2-IRES-NK4质粒转进减毒沙门菌Ty21a感受态,制备成重组的减毒沙门菌菌株TPIN;将TPIN在含有氨苄西林(+)和氨苄西林(-)的LB培养板传代培养至第10代、20代、30代和40代时用灭菌的牙签分别挑取含有氨苄西林(+)和氨苄西林(-)LB培养基上的单克隆菌落,质粒提取、PCR扩增酶切鉴定;TPIN转染HepG2细胞后采用RT-PCR检测IL-2和NK4基因表达,ELISA法检测细胞培养上清IL-2和NK4蛋白。结果构建菌株经提取质粒、酶切、PCR扩增获得目的基因IL-2、NK4特异条带;在氨苄西林(+)和氨苄西林(-)LB培养板传代培养40代的TPIN菌株均可扩增并双酶切出目的基因IL-2及NK4;TPIN体外转染HepG2细胞后,IL-2及NK4表达水平均显著升高。结论重组携带IL-2/NK4双基因的减毒沙门菌菌株TPIN可稳定遗传,不受无选择性压力的影响而丢失质粒,且在体外IL-2、NK4基因及蛋白可以稳定高效表达。
Objective To prepare an attenuated Salmonella typhimurium DNA vaccine containing pCMV-IL-2-IRES-NK4 plasmid (TPIN) and to detect its stability. Methods The pCMV-IL-2-IRES-NK4 plasmid was transformed into an attenuated Salmonella typhimurium Ty21a competence, and then a recombinant attenuated Salmonella typhimuri-um strain TPIN was prepared; the recombinant strain was cultivated in 40 generations on LB nutritional medium plate with or without Ampicillin, and monoclonal bacterial colony was selected at subculture 10th , 20th , 30th and 40th generation from Ampicillin ( + ) and no Ampicillin ( - ) plates respectively, and then the plasmids were extracted and identified by PCR amplification and enzyme digestion; TPIN was transfected into HepG2 cells, then expressions of the IL-2 / NK4 genes were determined by reverse transcription polymerase chain reaction (RT-PCR), and expressions of IL-2 / NK4 albumen cells were detected by enzyme linked immunosorbent assay (ELISA). Results Goal genes IL-2 and NK4 were obtained by extraction, enzyme digestion and PCR amplification; goal genes IL-2 and NK4 were obtained in 40 generations TPIN on both Ampicillin ( + ) and Ampicillin ( - ) of LB nutritional medium plate by enzyme digestion and PCR amplification;levels of IL-2 and NK4 were significantly increased after TPIN was transfected into HepG2 cells in vitro. Conclusion An attenuated Salmonella typhimurium strain (TPIN) containing IL-2 / NK4 expression vector pCMV-IL-2-IRES-NK4 can stably construct, and the plasmids can pass stably in spite of the selective pression, and IL-2 / NK4 genes can be efficient-ly expressed in vitro.
出处
《解放军医药杂志》
CAS
2014年第7期33-38,共6页
Medical & Pharmaceutical Journal of Chinese People’s Liberation Army
基金
甘肃省科技重大专项(1302FKDA039)
兰州市科技项目(2011-2-60)
全国博士后基金(20060390192)