摘要
目的构建表达结核分枝杆菌分泌蛋白Ag85B的靶细胞以便评定结核疫苗的特异性细胞毒(CTL)作用。方法将携带有Ag85B基因的重组质粒pTB30s转入小鼠骨髓瘤细胞(SP2/0),用RT-PCR及免疫组化染色法鉴定阳性克隆细胞胞内的Ag85B基因转录和蛋白表达水平;同时,用接种过BCG,pTB30s或生理盐水(NS)小鼠脾脏单个核细胞作为效应细胞,G418高压筛选的阳性克隆细胞为靶细胞,用MTT法检测各组CTL杀伤活性。结果RT-PCR以及免疫组化结果显示,阳性细胞能稳定表达Ag85B。CTL杀伤实验:BCG、pTB30s及NS对照组杀伤率分别为28.14%、45.18%和5.13%。结论含有Ag85B靶细胞构建成功及应用,为研究结核疫苗的免疫效果提供了较好的研究手段。
To construct the target cells that carry Ag85B antigen of Mycobacterium tuberculosis for the evaluation of the function of cytotoxic T lymphocytes （CTL） induced by vaccine for tuberculosis, the recombinant plasmid pTB30s carrying ag85B gene was transfected to mouse myeloma cell line SP2/0 cells with lipofectamine, and the positive clones were cloned with high concentration of G418. The transcription and expression levels of ag85B gene were identified by RT-PCR and immunohistochemical stain. Meanwhile, the cytotoxic activities of CTLs were assayed in various groups of mice by means of MTT assay, in which the splenic mononucleated cells of mice immunized with BCG, pTB30s or normal saline were used as effector cells, and the positive clone cells screened with high concentration of G418 were used as target cells. The experimental results showed that the positive clones screened could express stably the Ag85B antigen as demonstrated by RT-PCR and immunohistochemical staining. The cytotoxic rates of the effector cells on the target cells in group of mice immunized with BCG, pTB30s and the control group of mice with injection of saline were 28.14%, 45.18% and 5.13% respectively. It is evident that the target cells carrying Ag65B antigen was successfully constructed, by which it can be used to evaluate the function of CTLs induced by tuberculosis vaccine.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第3期214-217,共4页
Chinese Journal of Zoonoses
基金
重庆市教委资助项目(渝教科〔2004〕12号文)
重庆医科大学科技创新资助项目(CX200204)
