幽门螺杆菌塑性区hp4565基因的克隆表达及其功能初探  

The cloning and expressing of hp4565 gene and the preliminary study on its biological function in Helicobacter Pylori's plasticity zone

作　　者：付启云[1] 邵世和[2] 薛玉芹[3] 郑绍同[1] 

机构地区：[1]南京医科大学附属淮安第一医院检验科,江苏淮安223300 [2]江苏大学医学院病原生物学研究室,江苏镇江212013 [3]金坛市人民医院检验科,江苏常州213200

出　　处：《现代预防医学》2015年第15期2770-2773,共4页Modern Preventive Medicine

基　　金：国家自然科学基金项目(81271795)

摘　　要：目的为克隆表达幽门螺杆菌(Helicobacter pylori,H.pylori)临床株的塑性区hp4565基因,并制备抗体,以探讨其生物学功能。方法以标准株NCTC shfi470的hp4565基因组全长设计引物,以H.pylori临床株为模板,PCR扩增临床株中hp4565基因,经原核表达后,选定最适条件大量诱导表达并经Ni2+-NTA柱纯化的目的蛋白。用MTT法检测重组蛋白对胃上皮细胞GES-1增殖影响,定磷法检测重组蛋白ATP酶活力。用纯化蛋白为抗原免疫家兔后制备多克隆抗体,ELISA检测抗体效价。结果成功克隆表达了hp4565基因,全长942 bp,编码313个氨基酸,原核表达后SDS-PAGE电泳显示有41 k Da的融合蛋白,Western blot鉴定是预期HP4565蛋白。纯化后获得较高纯度的重组蛋白,MTT法结果显示HP4565蛋白在低浓度(≤40 mg/L)时对细胞增殖无明显影响;蛋白浓度在60 mg/L以上时,细胞增殖随着培养时间的延长和浓度的增加呈现逐渐降低的趋势;重组蛋白具有一定的ATP酶活力,免疫家兔制备的多克隆抗体效价为1∶1.6×105。结论成功克隆表达了H.pylori临床株塑性区hp4565基因,得到纯度较高的蛋白和滴度较高的抗体,重组蛋白对胃上皮细胞增殖有一定影响,并具有一定的ATP酶活力。Objective To clone and express hp4565 of Helicobacter Pylori（H pylori） clinical strains, and produce antibodies, so as to explore their biological function. Methods The primers came from hp4565 gene of NCTCshfi470, the templates came from the clinical strains, and hp4565 gene fragments were amplified by PCR technology. After the prokaryotic expression, the optimum condition was selected to induce and express a lot of proteins, which were purified by Ni2＋-NTA columns. The proliferation inhibition activity of HP4565 was detected by MTT method, and the ATPase activity was detected by testing P. The polyclonal antibodies were produced by immunization rabbits. At last, ELISA was used for detecting polyclonal antibodies titers. Results The hp4565 gene was cloned and expressed successfully and the full-length was 942 bp, with encoding 313 amino acids. After the prokaryotic expression, SDS-PAGE showed a new band protein was found in the 41 k Da position and it was HP4565 protein detected by Western Blot method. The better purity recombinant proteins could be obtained after purifying. We found that the protein at low concentrations（40mg/L） was no significance influence on GES-1 cells, while over 60mg/L, the protein inhibited cell proliferation with increment of the concentration and culture time. The recombinant proteins had some ATPase activity. The titers of polyclonal antibody were 1： 1.6×105, which produced by immunization rabbits. Conclusion The hp4565 of H pylori which came from clinical strains was cloned and expressed successfully, and the better purity recombinant proteins and higher titers of antibody were obtained. The recombinant proteins had influence on proliferation of gastric epithelial cell, and they had some ATPase activities.

关 键 词：幽门螺杆菌 基因 克隆表达 细胞增殖 ATP酶 

分 类 号：R117[医药卫生—公共卫生与预防医学]