结核杆菌cfp10真核载体的构建、表达与基因免疫

目的: 构建以结核分枝杆菌H37Rv cfp10基因为基础的基因疫苗. 方法: 采用BamH I和Xba I双酶切质粒pGEM-Teasy-CFP10中,10 g*L-1琼脂糖凝胶电泳回收约300 bp大小片段,以亚克隆法构建于真核表达载体pcDNA3.1的相应酶切位点. 阳性克隆提取质粒,体外电穿孔转化CHO细胞,RT-PCR法检测cfp10特异的mRNA的表达. 质粒肌注免疫BALB/c小鼠,ELISA检测小鼠血清中相应抗体的滴度. 结果: 用BamH I和XbaⅠ酶切鉴定证实目的基因正确插入载体pcDNA3.1,命名为pcCFP10; pcCFP10转化的CHO细胞RNA提取物中,RT-PCR扩增出300 bp大小的条带. 肌注免疫BALB/c小鼠,ELISA检测小鼠血清中相应抗体平均滴度为1∶ 4,000. 结论: 以CFP10的编码基因为基础的真核表达质粒的构建成功.

Construction,expression and gene immunization of eukaryotic expression vector containing cfp10 of Mycobacterium tuberculosis

AIM: To construct DNA vaccine based on cfp10 gene of mycobacterium tuberculosis H37Rv strain. METHODS: Plasmid pGEM-Teasy-CFP10 was digested by BamH Ⅰ and XbaⅠ and a fragment about 300 bp was recovered by 10 g*L-1 agarose electrophoresis and was subcloned to the responding sites of eukaryotic expression vector pcDNA3.1. Positive recombinant plasmid was transfected into Chinese hamster ovary (CHO) cells in vitro by electroporation and specific mRNA of cfp10 in RNA of transfected CHO cells was detected by RT-PCR method. BALB/c mice were immunized at two-week intervals by recombinant plasmid and specific antibody against CFP10 was detected by ELISA. RESULTS: The gene encoding CFP10 protein was correctly inserted into the vector pcDNA3.1 as confirmed by restriction endonuclease digestion and the positive plasmid we named pcCFP10. After pcCFP10 was transfected into CHO cells, about 300 bp size specific mRNA of cfp10 was detected by RT-PCR. Mean antibody titer of sera of BALB/c mice immunized with pcCFP10 was 1∶ 4,000. CONCLUSION: Recombinant eukaryotic expressing plasmid has been successfully constructed based on the gene encoding CFP10 of mycobacterium tuberculosis, which is helpful in the further study of immunological characteristics of protein CFP10 and in the prevention of tuberculosis.