CAJ | 학술논문

目的构建肺炎链球菌(Streptococcus pneumoniae,Sp)溶血素基因(pneumolysin,ply)缺陷菌株,并对其毒力作初步研究,为进一步探索宿主对溶血素的防御应答奠定基础.方法采用长臂同源多聚酶链反应(LFH-PCR)技术将ply基因替换为红霉素耐药基因(erm)后同源重组于肺炎链球菌,在含红霉素的血平板上筛选出ply缺陷菌株.用PCR鉴定缺陷菌株,观察体外缺陷菌株生长情况,并在小鼠体内感染模型研究其毒力侵袭变化.结果 PCR结果显示ply基因完全被erm基因所替代,构建ply缺陷菌成功;单个菌落培养基生长情况表明ply基因缺陷并未对细菌的体外生长造成影响;但在小鼠鼻腔感染模型中,缺陷菌株入血时间(6 h)明显晚于野生菌株(2 h),且各时间点的菌量均显著低于野生菌株,两者比较差异有统计学意义(P＜0.01);小鼠腹膜感染模型显示野生菌株半数致死时间为3 d,而缺陷菌株半数致死时间为18d,两者比较差异有统计学意义(P＜0.01).结论采用LFH-PCR技术作基因突变完全替代ply基因,方法简便快捷;ply的缺陷不影响细菌在体外的生长,但可显著降低细菌在宿主体内的毒力和侵袭.
목적 구건폐염련구균(Streptococcus pneumoniae,Sp)용혈소기인(pneumolysin,ply)결함균주,병대기독력작초보연구,위진일보탐색숙주대용혈소적방어응답전정기출.방법 채용장비동원다취매련반응(LFH-PCR)기술장ply기인체환위홍매소내약기인(erm)후동원중조우폐염련구균,재함홍매소적혈평판상사선출ply결함균주.용PCR감정결함균주,관찰체외결함균주생장정황,병재소서체내감염모형연구기독력침습변화.결과 PCR결과현시ply기인완전피erm기인소체대,구건ply결함균성공;단개균락배양기생장정황표명ply기인결함병미대세균적체외생장조성영향;단재소서비강감염모형중,결함균주입혈시간(6 h)명현만우야생균주(2 h),차각시간점적균량균현저저우야생균주,량자비교차이유통계학의의(P＜0.01);소서복막감염모형현시야생균주반수치사시간위3 d,이결함균주반수치사시간위18d,량자비교차이유통계학의의(P＜0.01).결론 채용LFH-PCR기술작기인돌변완전체대ply기인,방법간편쾌첩;ply적결함불영향세균재체외적생장,단가현저강저세균재숙주체내적독력화침습.
Objective To lay the foundation for further exploration on parasitifer's defence reaction to pneumolysin through constructing ply gene-deletion strain of Streptococcus pneumoniae and researching on its virulence change. Methods A linker fragment with erm gene in middle and homologous upstream and downstream fragment of ply gene at both sides was prepared by long flanking homology-polymerase chain reaction(LFH-PCR). The linker fragment was transformed into Streptococcus pneumoniae. ply-deficient strain was then screened out from blood plate which contains erythromycin and identified by PCR. ply-deficient strain growth in vitro was observed and virulence change was observed through infecting mouse model. Results PCR results showed that ply gene was replaced completely by erm gene. The ply deficient strain was successfully constructed. The growth of single strain culture medium showed that ply genetic defect made no influence on bacterial's external growth. While in the mice nasal cavity infecting experiment, deficient strain enter into blood after 6 h from infecting which obviously slower than that did wild-type(2 h). And the number of bacteria at each point was much smaller than that of wild-type(P ＜0. 01 ). The mice peritonaeum infecting experiment showed that median lethal time of wild-type was 3 d, while that of deficient strain was 18 d(P＜0. 01). Conclusion It is a good way to completely substitute ply gene using LFH-PCR. ply deletion made no influence on baterial's growth in vitro, but it resulting in reduction of bacterial virulence in vivo.