结核分枝杆菌特异性分泌抗原克隆和融合表达及抗原性鉴定  

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

作　　者：娄培安[1] 章辉[2] 刘加彬[1] 王晓婷[2] 周艳秋[1] 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221006 [2]江苏省血吸虫病防治研究所

出　　处：《中国校医》2006年第1期1-6,共6页Chinese Journal of School Doctor

基　　金：2003~2004年度江苏省预防医学科研基金课题(Y200424);徐州市2004年度第五批科技计划项目(X2004541)

摘　　要：目的融合表达结核分枝杆菌分泌蛋白CFP10-ESAT-6或ESAT-6-CFP10,研究其免疫学特性,为结核病的血清学诊断提供物质基础。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克隆入改建的载体pET32c(+)linker前,CFP10克隆入linker后,构建ESAT-6-CFP10融合基因,分别转化大肠杆菌XL1-blue,抽提质粒,酶切鉴定;在大肠肝菌BL21中表达,通过Westernblot分析其抗原性。结果二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。Westernblot分析表明,融合蛋白与活动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-6二种蛋白的抗原性。本研究为rCFP10-ESAT-6或rESAT-6-CFP10融合蛋白在结核病血清学诊断的中应用奠定了基础。Objective To investigate the fused expression of secreted protein CFP 10-ESAT-6 or EAST-6- CFP 10 of Mycobacterium tuberculosis, and to study their immunological characteristics, and their potential for serodiagnosis of tuberculosis. 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 CFP10 and KSAT-6 genes were amplified by PCR reaction and cloned into pET32c （ ＋ ）-linker either ahead of or following linker. The recombinant CFP10-ESAT-6 or EAST-6-CFP10 fusion protein was expressed in E. coli BL21. Their antigenicities were confirmed by Western blot. The potential value of the fusion proteins was evaluated in the diagnosis of tuberculosis with sera of tuberculosis patients. Results The sequence of recombinant plasmid pET32c （＋） CFP10-ESAT-6 or pET32c （＋）-EAST-6-CFP10 was identical to the predicted sequence. The recombinant protein （rCFP10-ESAT 6 or rEAST-6-CFP10）, about 26 000, existed in the cytoplasm BL21. Western blot showed that the rCFP10-ESAT-6 or rEAST-6-CFP10 had good immuoreactivity with sera from patients with active tuberculosis. Conclusions Multi-antigen plasmid encoding CFP10 and ESAT-6 of Mycobacterium tuberculosis is constructed successfully. The fusion protein CFP 10 or ESAT- 6 or EAST- 6- CFP 10 is obtained, so that it can provide an experimental basis for diagnosis of tuberculosis in serology.

关 键 词：CFP-10蛋白(71—85) 结核杆菌 6-kDa早期分泌性抗原靶 重组蛋白 血清学检验 

分 类 号：R378.911[医药卫生—病原生物学] R392.11[医药卫生—基础医学]