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目的在原核表达系统融合表达结核分枝杆菌早期分泌性抗原靶ESAT-6和培养滤液蛋白CFP10的融合蛋白(rE6C)并纯化,测定抗原性和特异性。方法用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ESAT-6、CFP10基因。将ESAT-6克隆入改建的载体pET32c(﹢)的linker前,CFP10克隆入linker后,构建E6C融合基因,转化大肠杆菌XL1-blue,抽提质粒,酶切鉴定;在大肠杆菌BL21中表达,纯化rE6C蛋白,通过酶联免疫吸附测定(ELISA)法检测其抗原性和特异性。结果重组质粒pET32c(﹢)-ESAT-6-CFP10靶基因的测序结果与预计序列完全一致。携带重组质粒的菌株经诱导产生高水平的表达产物,纯化的表达产物具备较高的纯度、抗原性和特异性。结论pET32c(﹢)-ESAT-6-CFP10质粒在BL21菌中能高效表达,rE6C融合蛋白具有良好的抗原性和特异性,有望用于结核分枝杆菌感染的临床诊断。
Objective To purify and express the fusion protein (rE6C) of ESAT-6 and filtrate CFP10 in the prokaryotic expression system, and determine the antigenicity and specificity. Methods The pET32c (+) - linker was constructed by inserting a flexible “linker” into prokaryotic expression vector pET32c (+). PCR amplification of ESAT-6, CFP10 gene. After cloning ESAT-6 into the linker of the modified vector pET32c (+) and CFP10 into the linker, the E6C fusion gene was constructed and transformed into E. coli XL1-blue. The plasmid was extracted and identified by restriction enzyme digestion. The recombinant plasmid was expressed in E. coli BL21, The rE6C protein was purified and tested for its antigenicity and specificity by enzyme-linked immunosorbent assay (ELISA). Results The sequencing results of the target gene of recombinant plasmid pET32c (+) - ESAT-6-CFP10 were completely consistent with the expected sequence. Strains carrying the recombinant plasmids are induced to produce high levels of expression products, and the purified expression products have high purity, antigenicity and specificity. Conclusion The plasmid pET32c (+) - ESAT-6-CFP10 is highly expressed in BL21 strain. The rE6C fusion protein has good antigenicity and specificity and is expected to be used in the clinical diagnosis of Mycobacterium tuberculosis infection.