CAJ | 학술논문

目的对经API细菌鉴定条所证实的眼源性蜡样芽孢杆菌进行溶血素BL(HBL)基因鉴定并进行体外毒理性试验.方法根据溶血素结合亚基B、2个溶血亚基L1、L2三种组分的基因核苷酸序列,设计合成3对特异性引物,通过体系和条件优化,建立PCR检测方法,并对5株临床分离的眼源性蜡样芽孢杆菌进行检测分析；随机选取其中1株菌,利用盐析法提取溶血素,并以不同浓度剂量作用于绵羊红细胞、Vero细胞和Hela细胞,监测其对细胞的毒性；通过腹腔注射观察HBL对小鼠的致死作用,评估其毒力的强弱.结果 5株临床分离菌中,共检出3个基因；蜡样芽孢杆菌溶血素BL蛋白具有较强溶血活性,其溶血活性呈明显的时间-剂量依赖性；BL溶血素对Vero细胞和Hela细胞的作用所致的形态变化虽有不同,但结果均导致细胞死亡,其内容物释出；对小鼠的致死率同样存在时间-剂量依赖性,且2.0 HU/ml浓度以上的BL溶血素可使小鼠48 h的致死率达100％.结论 HBL具有较强的溶血活性,对临床分离的眼源性蜡样芽孢杆菌BL基因进行检测,3个基因均表达,为该菌感染后病情发展迅速、预后较差的临床表现提供了理论依据,同时为该病的快速诊断和分子流行病学调查提供了新的方法.
목적 대경API세균감정조소증실적안원성사양아포간균진행용혈소BL(HBL)기인감정병진행체외독이성시험.방법 근거용혈소결합아기B、2개용혈아기L1、L2삼충조분적기인핵감산서렬,설계합성3대특이성인물,통과체계화조건우화,건립PCR검측방법,병대5주림상분리적안원성사양아포간균진행검측분석；수궤선취기중1주균,이용염석법제취용혈소,병이불동농도제량작용우면양홍세포、Vero세포화Hela세포,감측기대세포적독성；통과복강주사관찰HBL대소서적치사작용,평고기독력적강약.결과 5주림상분리균중,공검출3개기인；사양아포간균용혈소BL단백구유교강용혈활성,기용혈활성정명현적시간-제량의뢰성；BL용혈소대Vero세포화Hela세포적작용소치적형태변화수유불동,단결과균도치세포사망,기내용물석출；대소서적치사솔동양존재시간-제량의뢰성,차2.0 HU/ml농도이상적BL용혈소가사소서48 h적치사솔체100％.결론 HBL구유교강적용혈활성,대림상분리적안원성사양아포간균BL기인진행검측,3개기인균표체,위해균감염후병정발전신속、예후교차적림상표현제공료이론의거,동시위해병적쾌속진단화분자류행병학조사제공료신적방법.
Objective To identify the hemolysin BL(HBL) gene from Bacillus cereus of patients with endophthalmitis comfirmed by API bacterial identification test strip,and detect its biological activity in vitro.Methods Three pairs of specific primers were designed according to the gene sequence of HBL(B,L1 and L2 components),then the PCR assay were established through condition optimization,and to further detect five Bacillus cereus strains isolated from clinical patients with endophthalmitis.HBL with biological activity was extracted by salt fractionation from a randomly selected strain,and a series of different concentrations of HBL were prepared and acted on sheep red blood cells(SRBC),Vero cells and Hela cells; virulence of HBL was also assessed through observating lethal effect of which on mice with intraperitoneal injection.Results Three genes of HBL were detected in all B.cereus strains from clinical patients; Strong hemolytic activity of HBL showed obvious time-and dose-dependent.In the study,we found the morphological changes of Vero and Hela cells caused by HBL were different,but cell death were the same result with contents released; Within 48 h after infection,lethality of HBL for mice showed 100％ with the concentration of more than 2.0 HU/ml,and was also in a time-and dose-dependent manner.Conclusion HBL isolated from B.Cereus had high hemolysis activity and low concentration.The expression of all BL genes provided a strong basis for the clinical feature of B.Cereus infection,which was developed rapidly and with a poor prognosis.It also provided a new method for rapid diagnosis and molecular epidemiology investigation in clinical.