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目的 :构建以结核分枝杆菌H3 7Rvcfp10基因为基础的基因疫苗 .方法 :采用BamHI和XbaI双酶切质粒pGEM Teasy CFP10中 ,10 g·L-1琼脂糖凝胶电泳回收约 30 0bp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BamHI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 30 0bp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 ,0 0 0 .结论 :以CFP10的编码基因为基础的真核表达质粒的构建成功 .
OBJECTIVE: To construct a gene vaccine based on the Mycobacterium tuberculosis H3 7Rvcfp10 gene.METHODS: A fragment of about 300 bp was recovered by 10 g · L-1 agarose gel electrophoresis with restriction endonuclease BamHI and XbaI digested plasmid pGEM Teasy CFP10, Subcloned into the eukaryotic expression vector pcDNA3.1 corresponding restriction sites.The positive clones were extracted plasmids, in vitro electroporation transformed CHO cells, cfp10 mRNA specific RT-PCR detection.Mice immunized BALB / c Mice and ELISA were used to detect the titers of the corresponding antibodies in the serum of mice.Results: The recombinant plasmid pcDNA3.1 was confirmed by restriction enzyme digestion with BamHI and XbaI, and inserted into the vector pcDNA3.1. The band of 300bp was amplified and BALB / c mice were intramuscularly immunized, the average titer of the corresponding antibodies in the serum of the mice was 1: 4, 0 0 0 by ELISA.Conclusion: Based on the coding gene of CFP10 Construction of nuclear expression plasmid was successful.