重组结核杆菌分泌融合蛋白活性鉴定及应用
Activity identity and application of recombinant Mycobacterium tuberculosis CFP10-ESAT-6 or EAST-6-CFP10 fusion protein
-
摘要:目的 研究融合表达结核分枝杆菌分泌蛋白CFP10-ESAT-6或ESAT-6-CFP10免疫学特性。方法 将一个柔性的氨基酸接头插入原核表达载体pET32c(+)中, 构建pET32c(+)-linker。PCR法扩增CFP10、ESAT-6基因。将CFP10克隆入改建的载体pET32c(+)的linker前, ESAT-6克隆入linker后, 构建CFP10-ESAT-6融合基因; 或将ESAT-6克隆入改建载体pET32c(+)的linker前, CFP10克隆入linker后, 构建ESAT-6-CFP10融合基因, 分别转化大肠埃希菌XL1-blue, 抽提质粒, 酶切鉴定; 在大肠埃希菌BL21中表达, 通过Westernblot分析其抗原性。结果 2个目的基因分别被按顺序成功克隆入载体pET32c(+)的linker前或后。重组质粒pET32c(+)-CFP10-ESAT-6或pET32c(+)-ESAT-6-CFP10靶基因的测序结果与预计序列完全一致。融合蛋白在BL21菌中高效表达。Western blot分析表明, 融合蛋白与活性肺结核患者血清能发生特异性免疫反应。结论 成功地构建了多抗原基因DNA质粒; pET32c(+)-CFP10-ESAT-6或pET32c(+)-ESAT-6-CFP10质粒在BL21菌中能高效表达rCFP10-ESAT-6或rESAT-6-CFP10融合蛋白, 该蛋白兼具CFP10和ESAT-6两种蛋白的抗原性。Abstract:Objective To study the immunological characteristics of the secreted proteins CFP10-ESAT-6 or EAST-6- CFP10 of Mycobacterium tuberculosis by fused expression.Methods Apair of oligodeox ynucleotide named linker encoding 12 glycines and 3 serines was synthesized and cloned into plasmid pET32c(+)to construct pET32c(+)-linker.The CF P10 and ESAT-6 gene were amplified by PCR reaction and cloned into pET32c(+)-linker either ahead of or follow ing linker.the recombinant CFP10-ESAT-6 or EAST-6-CFP10 fusion protein was expressed in E.coli.BL21.Their antig enicity were confirmed by western blot.Results The sequence of recombinant plasmid pET32c(+)-CFP10-ESAT-6 or pET32c(+)-EAST- 6-CFP10 was identical to the predicted sequence.The recombinant proteins(rCFP10-ESAT-6 or rEAST-6-CFP10)can expressed effectively in the cytoplasm BL21.Western blot showed that the rCFP10-ESAT-6 or rEAST-6-CFP10 had good immuoreactivity with sera from patients with activetuberculosis.Conclusion Multi-antigen plasmid encoding CFP10 and ESAT-6 of Mycobacterium tuberculosis was constructed successfully.The fusion protein CFP10-ESAT-6 or EAST-6-CFP10 was obtained.The fusion proteins have antigenicity of EAST-6 and CFP10.
下载: