摘要
目的克隆表达结核分枝杆菌CFP10-ESAT6-Rv3425基因片段融合蛋白并纯化,测抗原的免疫原性,为结核病的临床诊断提供有效的候选抗原。方法利用生物信息学分析免疫优势位点,设计不同引物,采用聚合酶链反应(PolymeraseChain Reaction,PCR)扩增出相应基因片段,插入pET30a表达载体,转入大肠杆菌BL21(DE3),IPTG诱导表达目的蛋白,亲和纯化后用H37Rv免疫的兔血清进行间接酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定其免疫原性。结果 PCR扩增的CFP10、ESAT6和Rv33425基因片段与基因文库(Genbank)报道的完全一致。三者在大肠杆菌BL21(DE3)中融合表达的产物约为22kDa,与预计大小相吻合。Ni-NTA亲和纯化出目的蛋白。ELISA显示该蛋白具有较强的免疫原性。结论结核分枝杆菌CFP10-ESAT6-Rv3425融合基因片段的融合表达纯化后,可作为结核病免疫诊断的一个候选抗原。
Objective To investigate the fused expression of CFP10-ESAT6-Rv3425 gene fragment of Mycobacterium tuberculosis,and to provide a candidate antigen for TB serodiagnosis.Methods The an immunodominant epitopes were analyzed by means of bioinformatics,then were amplified by PCR They was cloned into corresponding sites of the expression vector pET30a and transformed into expressive strain E.coli BL21（DE3）,induced with IPTG and fusion protein was purified by Ni-NTA purification system.The specific antibody in the sera of rabbit immunized with the fused protein was detected by ELISA.Results The sequences by PCR amplification were identical to those reported by Genebank.The recombinant plasmid fused expression protein of CFP10-ESAT6-Rv3425 gene fragment with relative molecular mass（Mr） of 22kDa.It could be purified by Ni-NTA purification system.The specific antibody in the sera of rabbit immunized with H37Rv reacted with the fused protein.Conclusion soluble protein CFP10-ESAT6-Rv3425 gene fragment fused of Mycobacterium tuberculosis was successfully fused expressed in E.coli BL21（DE3）,it may become a novel candidate serodiagnosistic antigen against tuberculosis.
出处
《临床肺科杂志》
2011年第10期1533-1535,共3页
Journal of Clinical Pulmonary Medicine
基金
国家科技重大专项(NO.2008ZX10003003)
