[重组结核分枝杆菌CFP10-ESAT-6融合蛋白的制备与应用]

[Preparation and application of recombinant Mycobacterium tuberculosis CFP10-ESAT-6 fusion protein].

作者信息

Chen Quan, Zhu Dao-yin, Luo Xu-dong, Jiang Ying, Jiang Shan

机构信息

Department of Microbiology, Chongqing University of Medical Science, Chongqing 400016, China.

出版信息

Zhonghua Jie He He Hu Xi Za Zhi. 2004 Apr;27(4):244-8.

PMID:15144615

Abstract

OBJECTIVE

To prepare the recombinant CFP10-ESAT-6 fusion protein, and to study its immunological characteristics, and its potential for serodiagnosis of tuberculosis.

METHODS

The lhp-ESAT-6 fusion gene was amplified by Gene SOEing, and then cloned into pQE30 plasmid. The recombinant CFP10-ESAT-6 fusion protein was expressed and purified. Its antigenicity was confirmed by Western blot. Animal models infected with M. tuberculosis H(37)Rv strain and M. bovis BCG respectively were made to evaluate the potential value of the fusion protein in the serodiagnosis of tuberculosis.

RESULTS

The sequence of recombinant plasmid pQE30-CFP10-ESAT-6 was identical to the predicted sequence. The recombinant protein (rCFP10-ESAT-6), about 26 000, existed in the cytoplasm of DH5alpha in soluble form and represented 40% of the total bacterial protein. The purity and concentration of the final product was 98% and 1.2 g/L, respectively. Western blot showed that the rCFP10-ESAT-6 had good immunoreactivity with sera from patients with active tuberculosis and rabbits immunized with CFP10 and ESAT-6 respectively. The positive cutoff value was A(490) plus 2 standard deviation from negative guinea pig sera detected by ELISA. Serological reactivity to rCFP10-ESAT-6 was observed in 11 of the serum samples from guinea pigs with tuberculosis and 1 of sera from guinea pigs infected with BCG, while the serological reactivity to PPD was observed in 11 of sera from guinea pigs with tuberculosis and in 11 of sera from guinea pigs infected with BCG.

CONCLUSIONS

The rCFP10-ESAT-6 fusion protein was highly expressed in soluble form in E. coli. It had antigenicity of both CFP10 and ESAT-6, and could be used to differentiate infection with M. tuberculosis H(37)Rv strain from immunization with M. bovis BCG. The study provided experimental data for potential application of rCFP10-ESAT-6 in the diagnosis of tuberculosis.

摘要

目的

制备重组CFP10 - ESAT - 6融合蛋白，研究其免疫学特性及其在结核病血清学诊断中的应用潜力。

方法

采用基因拼接法（Gene SOEing）扩增lhp - ESAT - 6融合基因，然后克隆至pQE30质粒。表达并纯化重组CFP10 - ESAT - 6融合蛋白。通过蛋白质印迹法（Western blot）确认其抗原性。分别构建感染结核分枝杆菌H(37)Rv株和卡介苗的动物模型，以评估融合蛋白在结核病血清学诊断中的潜在价值。

结果

重组质粒pQE30 - CFP10 - ESAT - 6的序列与预测序列一致。重组蛋白（rCFP10 - ESAT - 6）分子量约为26 000，以可溶性形式存在于DH5α菌的细胞质中，占细菌总蛋白的40%。最终产物的纯度和浓度分别为98%和1.2 g/L。蛋白质印迹法显示，rCFP10 - ESAT - 6与活动性肺结核患者血清以及分别用CFP10和ESAT - 6免疫的兔血清具有良好的免疫反应性。阳性临界值为通过酶联免疫吸附测定（ELISA）检测的阴性豚鼠血清的A(490)加上2个标准差。11份结核病豚鼠血清样本和1份卡介苗感染豚鼠血清对rCFP10 - ESAT - 6有血清学反应，而11份结核病豚鼠血清和11份卡介苗感染豚鼠血清对结核菌素纯蛋白衍生物（PPD）有血清学反应。