肺炎链球菌组氨酸激酶VicK的全长表达及其保守性分析  

Expression and purification of Streptococcus pneumoniae full-length VicK protein and analysis of its conservation

作　　者：张帅[1] 刘宇思[1] 王虹[1] 王哲[1] 王维[1] 张雪梅[1] 

机构地区：[1]重庆医科大学检验系,临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国抗生素杂志》2014年第2期131-136,共6页Chinese Journal of Antibiotics

基　　金：重庆市科委自然科学基金针对TCS系统中YycG蛋白的抗菌中药先导化合物的高通量筛选(No.cstc2011jjA10063)

摘　　要：目的全长表达肺炎链球菌(Streptococcus pneumoniae,S.pn)VicK蛋白并制备其多克隆抗体,对其进行保守性分析和亚细胞定位,为其作为抗菌药物筛选靶点提供实验依据。方法利用分子生物学技术构建pET-28a(+)-vicK原核表达质粒,并在大肠埃希菌BL21(DE3)中进行表达,经Ni-NTA亲和纯化后,通过试剂盒检测其激酶活性。将纯化的VicK蛋白免疫Balb/C小鼠,并用ELISA和Western blot方法分别检测其多克隆抗体的效价及特异性。采用流式细胞术对VicK蛋白进行表面定位分析。结果成功获得VicK全长蛋白,酶活性检测结果提示具有ATP激酶活性。VicK蛋白免疫小鼠获得效价达1:125000以上的多克隆抗体。Western Blot结果显示其能特异地识别1型、2型、3型、4型、6B及19F型S.pn中的VicK蛋白。流式结果表明VicK蛋白主要位于胞内。结论获得具有ATP激酶活性的S.pn VicK全长蛋白,且该蛋白保守表达于S.pn胞内。Objective To obtain active Histidine kinase VicK protein of Streptococcus pneumonia and analysize its conservation and subcellular localization to provide experimental basis for the develoment of vick as an antibacterial drug target. Methods Recombinant ptasmid pET-28a（＋）-vicK was constructed using molecular biology techniques then transformed into BL21（DE3） and induced with IPTG. After purified by Ni-NTA affinity chromatography, the ATPase activity of the full-length VicK protein was detected by using Kinase-Glo Luminescent Kinase Assays. Then the VicK protein was introduced into BALB/C mice to obtain polyclonal antibody. Antibody titer and specificity were determined by ELISA and Western blot, respectively. Finally, the flow cytometry method was used for its subcellular localization. Results The soluble target VicK protein which has kinase activity ofhydrolyzing ATP in vitro was expressed Successfully in E.coli. antiserum was obtained after immunized with VicK protein. ELISA showed the titer of Antibody was up to 1：125000. The specificity of the antibody and the conservation of VicK protein in 6 different strains of Streptococcus pneumoniae was proved by Western blot. flow cytometry showed that VicK protein was mainly located in the intracellular. Conclusion We obtained active VicK protein which was mainly located in the intracellular and was conserved in Streptococcus pneumonia.

关 键 词：肺炎链球菌 VicK蛋白 表达纯化 多克隆抗体 激酶活性 定位 

分 类 号：R378.14[医药卫生—病原生物学]