[结核分枝杆菌四种特异性抗原的分子克隆、纯化及血清学特性分析]
[Molecular cloning, purification, and serological characterization of four specific antigens of Mycobacterium tuberculosis].
作者信息
Xu Lei, Li Hui-yun, Ma Li-heng
机构信息
Department of Biochemistry, Medical College, Tongji University, Shanghai 200092, China.
出版信息
Zhonghua Jie He He Hu Xi Za Zhi. 2004 Mar;27(3):188-90.
OBJECTIVE
To express the 14,000, 37,000, and 6,000 early secretory antigenic target (ESAT-6) and mtb81 antigen genes in bacteria, and to purify the product and determine their activity.
METHODS
The 14,000, 37,000 , ESAT-6, and mtb81 antigen genes were amplified from Mycobacterium tuberculosis genomic DNA by polymerase chain reactions and cloned into pGEX 4T-1 expression vector. BL21 strain of Escherichia coli was transformed with the recombinant vectors and induced to express recombinant proteins. The proteins were purified by affinity chromatography. The biological activity of purified proteins were estimated by enzyme-linked immunoabsorbant assay (ELISA).
RESULTS
The BL21 strains of Escherichia coli with recombinant vectors showed high level of 14,000, 37,000, ESAT-6, and mtb81 gene expressions after induction. The products were purified successfully and showed high antigenicity and specificity. The sensitivity of 38,000, 14,000, ESAT-6, and mtb81 were 54%, 60%, 44%, and 36%, respectively.
CONCLUSION
The expressions and purifications of recombinant 14,000, 37,000, ESAT-6, and mtb81 antigens with natural activity facilitate their research and application.
目的
在细菌中表达14kDa、37kDa早期分泌性抗原靶标（ESAT-6）和mtb81抗原基因，纯化产物并测定其活性。
方法
通过聚合酶链反应从结核分枝杆菌基因组DNA中扩增14kDa、37kDa、ESAT-6和mtb81抗原基因，并克隆到pGEX 4T-1表达载体中。用重组载体转化大肠杆菌BL21菌株并诱导表达重组蛋白。通过亲和层析纯化蛋白。用酶联免疫吸附测定（ELISA）评估纯化蛋白的生物学活性。
结果
诱导后，带有重组载体的大肠杆菌BL21菌株显示出高水平的14kDa、37kDa、ESAT-6和mtb81基因表达。产物成功纯化并显示出高抗原性和特异性。37kDa、14kDa、ESAT-6和mtb81的敏感性分别为54%、60%、44%和36%。
结论
具有天然活性的重组14kDa、37kDa、ESAT-6和mtb81抗原的表达和纯化有助于它们的研究和应用。
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