CAJ | 학술논문

目的：确定问号钩端螺旋体(简称钩体) LA2144基因产物血小板活化因子乙酰水解酶( PAF-AH)活性、感染细胞时表达与分泌情况,以及诱导金地鼠内脏出血的作用。方法采用PCR扩增问号钩体黄疸出血群赖型赖株无信号肽LA2144基因并构建其原核表达系统,采用Ni-NTA亲和层析法提纯表达的目的重组蛋白rLep-PAF-AH。采用分光光度法检测rLep-PAF-AH水解PAF底物的活性、水解效率及Km和Kcat值。采用实时荧光定量RT-PCR( qRT-PCR)和Western blot法分别检测问号钩体赖株感染人脐静脉血管内皮细胞( HUVEC)、人单核细胞THP-1和小鼠巨噬细胞J774A.1后,LA2144基因mRNA( Lep-PAF-AH-mRNA)水平变化及产物外分泌情况。金地鼠尾静脉两次注射100μg无LPS的rLep-PAF-AH后观察肺、肝、肾组织中出血情况。结果所构建的问号钩体赖株LA2144基因原核表达系统在IPTG诱导下能高效表达rLep-PAF-AH,Ni-NTA亲和层析法提纯的rLep-PAF-AH经SDS-PAGE 后显示为单一的蛋白条带。5μg rLep-PAF-AH 对底物的水解效率为26.6 U/L,其Km和Kcat值分别为82.79μmol/L和0.24 S-1。问号钩体赖株与HUVEC、THP-1和J774A.1细胞共培养1或2 h,Lep-PAF-AH-mRNA水平显著升高(P<0.05)。问号钩体赖株与上述细胞共培养2~12 h,培养物上清中检出大量Lep-PAF-AH,但其EMJH培养物上清中未检出Lep-PAF-AH。 rLep-PAF-AH注射金地鼠出现肺出血,但肝、肾组织中无明显出血现象。结论 LA2144基因产物具有较强PAF-AH活性,感染细胞时表达上调并外分泌、诱导金地鼠肺出血,故是问号钩体感染时引起宿主出血的重要毒力因子。
목적：학정문호구단라선체(간칭구체) LA2144기인산물혈소판활화인자을선수해매( PAF-AH)활성、감염세포시표체여분비정황,이급유도금지서내장출혈적작용。방법채용PCR확증문호구체황달출혈군뢰형뢰주무신호태LA2144기인병구건기원핵표체계통,채용Ni-NTA친화층석법제순표체적목적중조단백rLep-PAF-AH。채용분광광도법검측rLep-PAF-AH수해PAF저물적활성、수해효솔급Km화Kcat치。채용실시형광정량RT-PCR( qRT-PCR)화Western blot법분별검측문호구체뢰주감염인제정맥혈관내피세포( HUVEC)、인단핵세포THP-1화소서거서세포J774A.1후,LA2144기인mRNA( Lep-PAF-AH-mRNA)수평변화급산물외분비정황。금지서미정맥량차주사100μg무LPS적rLep-PAF-AH후관찰폐、간、신조직중출혈정황。결과소구건적문호구체뢰주LA2144기인원핵표체계통재IPTG유도하능고효표체rLep-PAF-AH,Ni-NTA친화층석법제순적rLep-PAF-AH경SDS-PAGE 후현시위단일적단백조대。5μg rLep-PAF-AH 대저물적수해효솔위26.6 U/L,기Km화Kcat치분별위82.79μmol/L화0.24 S-1。문호구체뢰주여HUVEC、THP-1화J774A.1세포공배양1혹2 h,Lep-PAF-AH-mRNA수평현저승고(P<0.05)。문호구체뢰주여상술세포공배양2~12 h,배양물상청중검출대량Lep-PAF-AH,단기EMJH배양물상청중미검출Lep-PAF-AH。 rLep-PAF-AH주사금지서출현폐출혈,단간、신조직중무명현출혈현상。결론 LA2144기인산물구유교강PAF-AH활성,감염세포시표체상조병외분비、유도금지서폐출혈,고시문호구체감염시인기숙주출혈적중요독력인자。
Objective To analyze the platelet activating factor acetylhydrolase ( PAF-AH) activity of a gene product encoded by LA2144 gene of Leptospira interrogans ( L. interrogans) , to investigate the ex-pression and secretion of LA2144 protein in various cell cultures and to further understand its function in in-ducing internal hemorrhage in an animal model. Methods The DNA sample containing LA2144 gene was extracted from L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai and used as the template for gene cloning by PCR. The LA2144 gene without the signal sequence coding region was amplified by PCR and inserted into a prokaryotic expression construct for the protein expression. The expressed recombinant protein, rLep-PAF-AH, was purified by Ni-NTA affinity chromatography. Spectrophotometry was used to measure the hydrolytic activity, hydrolytic efficiency, Km and Kcat values of the rLep-PAF-AH protein in hydrolyzing PAF substrate. Real-time fluorescent quantitative RT-PCR ( qRT-PCR) and Western blot assay were performed to measure the expression of LA2144 gene at mRNA and protein levels in human umbilical vein endothelial cells (HUVEC), human monocytes (THP-1) and murine macrophages (J774A. 1) with L. interrogans strain Lai infection, respectively. Each syrian hamster was intravenously injected with 100 μg of LPS-free rLep-PAF-AH for two times. Hemorrhage in the lungs, livers and kidneys were observed in three days after the injection. Results The constructed prokaryotic expression system for LA2144 gene of L. inter-rogans strain Lai could highly express the rLep-PAF-AH upon the induction of IPTG. The purified rLep-PAF-AH showed high purity with a single protein band in gel as indicated by SDS-PAGE. The efficiency of 5 μg of rLep-PAF-AH in hydrolyzing PAF substrate was 26. 6 U/L with a Km value of 82. 79 μmol/L and a Kcat value of 0. 24 S-1 . The expression of Lep-PAF-AH at mRNA level in HUVEC, THP-1 and J774A. 1 cells were significantly elevated after co-culture with L. interrogans strain Lai for 1 or 2 hours (P<0. 05). A large amount of Lep-PAF-AH were detected in the supernatants from co-cultures of L. interrogans strain Lai with the three cell lines, but not from the culture of the spirochete in EMJH medium. The signs of hemor-rhage were observed in the lung of hamsters injected with rLep-PAF-AH, but not in tissue samples from liver and kidney. Conclusion The LA2144 gene product was characterized by a stronger PAF-AH activity. The expression of LA2144 gene at mRNA and protein levels in various cell lines were enhanced during L. interro-gans infection. Moreover, the rLep-PAF-AH could induce the pulmonary hemorrhage in hamsters. This stud-y indicated that the protein encoded by LA2144 gene was an important virulence factor causing hemorrhage in hosts during L. interrogans infection.