中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
6期
523-527
,共5页
丁士标%林旭瑷%王欢%严杰
丁士標%林旭璦%王歡%嚴傑
정사표%림욱애%왕환%엄걸
问号钩端螺旋体%sph2基因/重组表达/瞬时表达%鞘磷脂酶类溶血素%溶血活性%细胞凋亡
問號鉤耑螺鏇體%sph2基因/重組錶達/瞬時錶達%鞘燐脂酶類溶血素%溶血活性%細胞凋亡
문호구단라선체%sph2기인/중조표체/순시표체%초린지매류용혈소%용혈활성%세포조망
Leptospira interrogans%sph2 gene/recombinant expression/transient expression%Sphingomyelinase hemolysin%Hemolytic activity%Cell apoptosis
目的 了解问号钩端螺旋体(简称钩体)感染细胞前后sph2基因表达水平变化,确定鞘磷脂酶类溶血素Sph2及诱导细胞凋亡的活性.方法 采用PCR从黄疸出血群赖型赖株钩体基因组DNA中扩增全长sph2基因片段,T-A克隆后测序.构建sph2基因原核表达系统,采用SDS-PAGE检查重组Sph2(rSph2)的表达情况,Ni-NTA亲和层析法提纯rSph2.采用绵羊血平板溶血试验及血红蛋白分光光度法测定rSph2的溶血活性.采用流式细胞术检测rSph2诱导小鼠单核-巨噬样细胞株J774A.1和肝细胞株IAR20凋亡的活性,实时荧光定量PCR检测赖株钩体感染J774A.1和IAR20细胞前后sph2基因mRNA水平变化.结果 与GenBank中sph2基因比较,所克隆的sph2基因序列相似性为100%.所构建的原核表达系统能高效表达rSph2.rSph2以浓度依赖方式溶解绵羊红细胞.10 μg/ml rSph2可诱导J774A.1和IAR20细胞凋亡,凋亡率峰值分别为23.96%和32.92%.赖株钩体感染J774A.1和IAR20细胞后0.5～2 h内sph2基因mRNA水平显著升高,2 h后mRNA水平迅速下降.结论 钩体sph2基因呈宿主细胞接触式瞬时表达.rSph2有溶解绵羊红细胞及诱导巨噬细胞和肝细胞凋亡的活性,因而Sph2是钩体致病过程中重要的毒力因子.
目的 瞭解問號鉤耑螺鏇體(簡稱鉤體)感染細胞前後sph2基因錶達水平變化,確定鞘燐脂酶類溶血素Sph2及誘導細胞凋亡的活性.方法 採用PCR從黃疸齣血群賴型賴株鉤體基因組DNA中擴增全長sph2基因片段,T-A剋隆後測序.構建sph2基因原覈錶達繫統,採用SDS-PAGE檢查重組Sph2(rSph2)的錶達情況,Ni-NTA親和層析法提純rSph2.採用綿羊血平闆溶血試驗及血紅蛋白分光光度法測定rSph2的溶血活性.採用流式細胞術檢測rSph2誘導小鼠單覈-巨噬樣細胞株J774A.1和肝細胞株IAR20凋亡的活性,實時熒光定量PCR檢測賴株鉤體感染J774A.1和IAR20細胞前後sph2基因mRNA水平變化.結果 與GenBank中sph2基因比較,所剋隆的sph2基因序列相似性為100%.所構建的原覈錶達繫統能高效錶達rSph2.rSph2以濃度依賴方式溶解綿羊紅細胞.10 μg/ml rSph2可誘導J774A.1和IAR20細胞凋亡,凋亡率峰值分彆為23.96%和32.92%.賴株鉤體感染J774A.1和IAR20細胞後0.5～2 h內sph2基因mRNA水平顯著升高,2 h後mRNA水平迅速下降.結論 鉤體sph2基因呈宿主細胞接觸式瞬時錶達.rSph2有溶解綿羊紅細胞及誘導巨噬細胞和肝細胞凋亡的活性,因而Sph2是鉤體緻病過程中重要的毒力因子.
목적 료해문호구단라선체(간칭구체)감염세포전후sph2기인표체수평변화,학정초린지매류용혈소Sph2급유도세포조망적활성.방법 채용PCR종황달출혈군뢰형뢰주구체기인조DNA중확증전장sph2기인편단,T-A극륭후측서.구건sph2기인원핵표체계통,채용SDS-PAGE검사중조Sph2(rSph2)적표체정황,Ni-NTA친화층석법제순rSph2.채용면양혈평판용혈시험급혈홍단백분광광도법측정rSph2적용혈활성.채용류식세포술검측rSph2유도소서단핵-거서양세포주J774A.1화간세포주IAR20조망적활성,실시형광정량PCR검측뢰주구체감염J774A.1화IAR20세포전후sph2기인mRNA수평변화.결과 여GenBank중sph2기인비교,소극륭적sph2기인서렬상사성위100%.소구건적원핵표체계통능고효표체rSph2.rSph2이농도의뢰방식용해면양홍세포.10 μg/ml rSph2가유도J774A.1화IAR20세포조망,조망솔봉치분별위23.96%화32.92%.뢰주구체감염J774A.1화IAR20세포후0.5～2 h내sph2기인mRNA수평현저승고,2 h후mRNA수평신속하강.결론 구체sph2기인정숙주세포접촉식순시표체.rSph2유용해면양홍세포급유도거서세포화간세포조망적활성,인이Sph2시구체치병과정중중요적독력인자.
Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.
