Deshpande R G, Khan M B, Navalkar R G
Department of Microbiology and Immunology, Morehouse School of Medicine, Atlanta, Georgia 30310.
Tuber Lung Dis. 1993 Dec;74(6):382-7. doi: 10.1016/0962-8479(93)90081-8.
To purify and study the seroreactivity of native and recombinant 12-kilodalton protein of Mycobacterium tuberculosis H37Rv.
M. tuberculosis H37Rv cells and Escherichia coli XL-1 containing the plasmid PRL4 encoding the M. tuberculosis heat shock protein GroES homolog were used as sources for the purification of native and recombinant 12 kD of M. tuberculosis respectively. The seroreactivity of the 12 kDs was studied by ELISA using sera from 35 leprosy and 25 active pulmonary tuberculosis (TB) patients, and from 10 normal healthy controls.
The 12 kD protein was purified from H37Rv extract (s12 kD) and from recombinant E. coli (r12 kD) by ultrafiltration and MonoQ fast pressure liquid chromatography (FPLC). Analysis of s12 kD and r12 kD by SDS-PAGE revealed a single protein band in both cases with an approximate molecular weight of 12,000 which was recognized by monoclonal antibody SA-12 in immunoblotting. Both the proteins exhibited a pI of approximately 4.6 by isoelectric focusing. Both the 12 kD proteins exhibited 96% positivity with TB sera as compared to normal control sera (P < 0.01). Only one serum sample from the 35 leprosy sera tested exhibited binding to both the s12 kD and r12 kD proteins. Delayed type hypersensitivity reaction to the 12 kD proteins was elicited in guinea pigs that had been immunized with H37Rv sonicate.
The 12 kD protein could be easily purified and could serve as a valuable serodiagnostic tool in the screening of TB cases from a large population in an endemic area.
纯化并研究结核分枝杆菌H37Rv天然及重组的12千道尔顿蛋白的血清反应性。
分别以结核分枝杆菌H37Rv细胞和含有编码结核分枝杆菌热休克蛋白GroES同源物的质粒PRL4的大肠杆菌XL-1作为纯化结核分枝杆菌天然及重组12 kD蛋白的来源。采用酶联免疫吸附测定（ELISA），利用35例麻风病患者、25例活动性肺结核患者以及10名正常健康对照者的血清，研究12 kD蛋白的血清反应性。
通过超滤和MonoQ快速压力液相色谱（FPLC）从H37Rv提取物（s12 kD）和重组大肠杆菌（r12 kD）中纯化出12 kD蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析s12 kD和r12 kD，两者均显示出一条单一蛋白条带，其近似分子量为12,000，在免疫印迹中可被单克隆抗体SA-12识别。等电聚焦显示两种蛋白的等电点均约为4.6。与正常对照血清相比，两种12 kD蛋白与肺结核血清的阳性反应率均为96%（P < 0.01）。在检测的35例麻风病血清中，只有1份血清样品与s12 kD和r12 kD蛋白均有结合。用H37Rv超声裂解物免疫的豚鼠对12 kD蛋白产生了迟发型超敏反应。
12 kD蛋白易于纯化，可作为在流行地区从大量人群中筛查肺结核病例的有价值的血清学诊断工具。
