单核细胞增多性李斯特菌hlyA基因序列及溶血素活性测定

Analysis of hlyA gene and hemolytic activity of Listeria monocytogenes from different source

从3株不同来源的单核细胞增多性李斯特菌中PCR扩增溶血素编码基因hlyA,连接到T克隆载体转化到受体菌DH5α中,经菌落PCR和提取质粒酶切鉴定后测序,对测定的hlyA基因核苷酸序列及推导的氨基酸序列进行分析,结果表明,从3个菌株克隆的hlyA基因具有完整的开放阅读框,同源性在96.9%以上;其推导的LLO氨基酸序列同源性在97.9%以上,N端存在相同的PEST基序,C端存在相同的保守性11肽结构。建立和运用分光光度法对3个试验菌株的溶血素活性进行了检测,结果表明,在pH5.6时LLO溶血活性最高,在pH7.0时LLO溶血活性基本丧失。本试验单核细胞增多性李斯特菌的流行病学分析和基因工程疫苗载体研究提供了依据。

Listeriolysin O （LLO） encoded by hlyA gene is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes. Total DNA extracted from 3 different Listeria monocytogenes strains （XFL0605, CCTCCAB97021 and C53004） and used to amplify the hlys by PCR,in which BamH Ⅰ and Ava Ⅰ/Xho Ⅰ sites were added at the 5′ and 3′ end of the PCR product respectively. PCR product was cloned into pTA2 directly and introduced into E. coli DH5α through transfeetion and positive clones were selected. The insert DNA was verified by enzyme digestion and DNA sequencing. The results showed that the nueleotide homology of them ranged from 96.9% to 100% ,and deduced amino acid sequence homology of the three strains more than 97.9%. LLO contains a same PEST-like sequence at the N terminus,and a same conserved undecapeptide, ECTGLAWEWWR,at the C terminus. The same time, the 1-24 amino acids were the signal peptide sequence properly,and digest site between the 24 and 25 sites. A simple and sensitive ultraviolet spectrophotometry method was established for determining the haemolytic activity of LM strains culture supernatants. The coefficient of correlation between SRBC condensity and D540 was 0.99 995. The HC50 values of XFL0605, CCTCCAB97021, and C53004 culture supernatants were 18. 85, 21.38,and19.68,show a clear correlationship to LLO concentration. It also shows a close relationship between haemolytic activity of the culture supernatants and the pH values, LLOs were active at pH5.6 and lost activity while pH value above 7. 0.