CAJ | 학술논문

目的原核表达肺炎链球菌毒力因子ClpP,探讨其作为肺炎链球菌候选蛋白疫苗的价值.方法分离培养TIGR4型肺炎链球菌,获取其染色体DNA,采用基因体外重组的方法将完整的ClpP开放读码框架克隆到pET-32a原核表达载体内,经测序鉴定、原核表达及纯化,ClpP主动和阳性抗体血清被动免疫小鼠,TIGR4型肺炎链球菌攻击后,监测其生存时间.结果获得了高表达的重组抗原蛋白,表达的抗原蛋白用Western blot鉴定,镍柱纯化并透析复性可得到纯度达90%以上的重组蛋白.主动和被动免疫BALB/c小鼠,与未免疫组相比,产生的保护性作用具有统计学意义.结论 ClpP蛋白免疫小鼠可抵抗肺炎链球菌侵袭性感染,ClpP蛋白可作为肺炎链球菌的候选蛋白疫苗.
목적 원핵표체폐염련구균독력인자ClpP,탐토기작위폐염련구균후선단백역묘적개치.방법 분리배양TIGR4형폐염련구균,획취기염색체DNA,채용기인체외중조적방법장완정적ClpP개방독마광가극륭도pET-32a원핵표체재체내,경측서감정、원핵표체급순화,ClpP주동화양성항체혈청피동면역소서,TIGR4형폐염련구균공격후,감측기생존시간.결과 획득료고표체적중조항원단백,표체적항원단백용Western blot감정,얼주순화병투석복성가득도순도체90%이상적중조단백.주동화피동면역BALB/c소서,여미면역조상비,산생적보호성작용구유통계학의의.결론 ClpP단백면역소서가저항폐염련구균침습성감염,ClpP단백가작위폐염련구균적후선단백역묘.
Objective To obtain purified ClpP produced by prokaryotic expression system,and to evaluate the protection effect elicited by ClpP in animal protection test.Methods The template DNA was isolated from the culture of TIGR4 Streptococcus pneumoniae.The complete ClpP open reading frame(ORF)was cloned into pET-32a expression vector by gene recombination technology in vitro.After prokaryotic expression,purification and sequence identification,the recombinant ClpP were innoculated into mice,and at the same time a group of mice were inoculated with the antibody to ClpP.We monitored the survival time of the innoculated mice after being challenged intraperitoneally with,TIGR4.Results We obtained high-expressed recombinant antigen protein which was then identified by Western blot,and we have got the recombinant antigen protein with a purity of more than 90%after being purified through Ni2+ affinity chromatography and processed by dialysis.The sunrvival time of mice immunized with ClpP or ClpP antibody were significantly langer than that of the mice received PBS.Conclusion The recombinant ClpP antigen protein can elicit protection to the invasive S.pneumoniae infection in mice,which might make ClpP as a candidate of S.pneumoniae vaccine.