结核分枝杆菌分泌蛋白ESAT-6-CFP10的融合表达及纯化  

Molecular cloning, fused expression and application of recombinant Mycobacterium tuberculosis ESAT- 6- CFP10 fusion protein

作　　者：娄培安[1] 王晓婷[2] 周艳秋[1] 章辉[2] 刘加彬[1] 李玲 赵广法[1] 朱荫昌[2] 

机构地区：[1]徐州市疾病预防控制中心,江苏徐州市221001 [2]江苏省血吸虫病防治研究所 [3]徐州市泉山区疾病预防控制中心

出　　处：《热带病与寄生虫学》2005年第4期196-200,195,共6页Journal of Tropical Diseases and Parasitology

基　　金：2003-2004年度江苏省预防医学科研基金项目(Y200424);徐州市2004年度第五批科技计划项目(x2004541)

摘　　要：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 express fused gene of 6000 early secretory antigenic target（ESAT-6） and culture filtrate protein 10 in bacteria,and to purify the product and determine their activity. Methods A pair of oligodeoxynucleotide named linker encoding 12 glycines and 3 serines was synthesized and cloned into plasmid pET32c（ ＋ ） to construct pET32c（ ＋ ）-linker. The ESAT-6 and CFP10 gene were amplified by PCR reaction and cloned into pET32c （ ＋ ）-linker. The recombinant E6C fusion protein was expressed in E.coli. BL21, Their antigenicity were confirmed by Western blot. The recombinant E6C fusion protein was purified by Ni-NTA purification system, The biological activity of purified protein were estimated by enzyme-linked immunoabsorbant assay （ELISA）. Results The sequence of recombinant plasmid pET32c （ ＋ ）- ESAT-6- CFP10 was identical to the predicted sequence. The recombinant protein（rE6C）,about 26 000, existed in the cytoplasm BL21 in soluble form. The pure product showed high antigenicity and specificity. The sensitivity and specificity were 71% and 100%, respectively. Conclusion The expression and purification of recombinant rE6C antigen with natural activity facilitate their research and application.

关 键 词：结核分枝杆菌 抗原 重组 酶联免疫吸附测定 

分 类 号：R52[医药卫生—内科学] R394.8[医药卫生—临床医学]