重组DNA
大肠杆菌
结核分枝杆菌
分子生物学
化学
克隆(编程)
亲和层析
分子质量
质粒
生物
生物化学
基因
酶
肺结核
医学
病理
计算机科学
程序设计语言
作者
B. Ramalingam,Alain R. Baulard,Camille Locht,P R Narayanan,Alamelu Raja
标识
DOI:10.1016/j.pep.2004.01.016
摘要
A limited number of proteins of Mycobacterium tuberculosis have been characterized so far for their use as potential candidates for diagnosis and vaccine studies. This study was aimed at cloning, expression, and purification of a 27 kDa protein (otherwise known as the MPT51 or Rv3803c protein) of M. tuberculosis. The Rv3803c gene was PCR amplified using primers that contain specific restriction sites. The amplified product was inserted initially into pTOPO and then sub-cloned into pET15b and pET24d vectors, such that the recombinant protein is predicted to contain an N-terminal or a C-terminal histidine tag, respectively. The recombinant plasmids were introduced into Escherichia coli BL21 (DE3) and the recombinant proteins were purified from the cytosolic fractions of the E. coli sonicates by nickel-NTA chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high performance liquid chromatography (HPLC), matrix assisted laser desorption-ionization-time-of-flight (MALDI-TOF), and circular dichroism (CD) studies, respectively. The purified proteins were found to be immunogenic and useful for immunodiagnostic studies of tuberculosis by enzyme linked immunosorbent assay (ELISA), with a sensitivity of 71% and specificity of 95%.
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