摘要
目的观察重组结核分枝杆菌RpfE蛋白对BCG的促生长作用。方法将测序正确的Rv2450c基因克隆到表达载体pET32a(+)中,获得RpfE蛋白,在变性条件下对目的蛋白进行亲和层析纯化。将纯化的蛋白分别与结核病人血清,Rpf单抗和Rpf结构域单抗进行Western blot分析。分别将10pmol/L和100pmol/L的RpfE加入到休眠BCG的培养基中,观察RpfE蛋白对BCG的促生长作用。结果得到融合组氨酸残基的重组RpfE蛋白,Western blot结果显示该蛋白分别与活动期结核病人血清,Rpf单抗和Rpf结构域单抗在40kDa处发生反应。100pmol/L的RpfE蛋白对BCG休眠菌具有明显的复苏和促生长作用。结论构建了MtbRv2450c基因的重组表达载体,纯化获得了RpfE重组蛋白,该蛋白对BCG休眠菌具有复苏和促生长作用。
To determine the exact role of recombinant RpfE protein of M. tuberculosis in resuscitationof dormant Bacille Calmette Guerin （BCG）, the sequenced Rv2450c gene （encoding RpfE） was cloned into pET32a （＋） vector and RpfE protein was expressed in Escherichia coll. The RpfE protein was purified with Ni-NTA purification system and the purified proteins＇ interaction with serum of tuberculosis patients, anti-Rpf antibody and anti-Rpf domain antibody were analyzed by Western blot respectively. Different concentrations of RpfE protein （10pmol/L and 100pmol/ID were added into culture medium of dormant BCG respectively and the resuscitation activity of RpfE protein to dormant BCG was observed. Results released that the recombinant RpfE protein which fused histidine residue was obtained. And the Western blot results showed that the protein＇s interaction with serum of tuberculosis patients, anti-Rpf antibody and anti Rpf domain antibody all located at 40kDa. Additionally, the resuscitation activity was remarkable when the concentration was 100pmol/L compared to 0pmol/L and 10pmol/L. Recombinant expression vector of Mtb Rv2450c gene was constructed and the purified recombinant RpfE protein was obtained. These results strongly suggest that the recombinant RpfE protein play a crucial role in resuscitation of dormant BCG.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2010年第10期919-922,共4页
Chinese Journal of Zoonoses
基金
国家"863"专项课题(2007AA02Z473)
国家自然科学基金(30972767)
陕西省自然科学基金(SJ08C203)联合资助
关键词
结核分枝杆菌
RpfE
休眠菌
促生长作用
Mycabacteriurn tuberculosis
RpfE
dormant BCG
resuscitation activity
