摘要
目的确定不同嗜肺军团菌lvgA基因序列的差异性及其感染宿主细胞后转录水平的变化,建立基于lvgA基因检测嗜肺军团菌的荧光定量RT-PCR。方法通过使用NCBI/Blast,Pfam,TMHMMServer v.2.0等网络资源分析嗜肺军团菌lvgA基因编码蛋白的保守功能结构域、跨膜区域。根据参考序列设计引物,采用PCR技术从嗜肺军团菌基因组DNA中扩增lvgA基因,将其连接入克隆载体pMD18-T进行测序,并用cltust软件比较不同嗜肺军团菌菌株lvgA基因的核苷酸、氨基酸序列。根据lvgA基因和嗜肺军团菌16SrDNA序列内的特异保守区域设计引物,以实时荧光定量RT-PCR检测嗜肺军团菌在感染人单核巨噬样细胞THP-1后lvgA基因转录水平的变化,同时,采用基于lvgA基因的荧光定量RT-PCR对嗜肺军团菌患者血液样本进行检测。结果研究表明,嗜肺军团菌lvgA基因为军团菌毒力基因,其产物定位于外膜表面。不同嗜肺军团菌菌株中lvgA基因的核苷酸序列和氨基酸序列保守,相似度分别达:99%,96%,96%和99%,98%,98%。在感染宿主细胞后lvgA基因的转录水平上调明显,最高达4.3倍(P<0.05)。基于lvgA基因的荧光定量RT-PCR方法可有效地检测嗜肺军团菌患者血液样本中的嗜肺军团菌。结论 lvgA基因与嗜肺军团菌的毒力相关,建立的基于lvgA基因的荧光定量RT-PCR方法具有快速、敏感、稳定等优点,适用于嗜肺军团菌肺炎临床实验室的早期诊断。
To analyse the transcription level alteration of lvgA gene in Legionella pneumophila after infecting host cells and establish a real-time fluorescent quantitative RT-PCR to detect L.pneumophila based on virulence gene（lvgA）.The structure domains,transmembrane regions have been analyzed through using on-line database of NCBI,Pfam and TMHMM Server v.2.0.The primers have been worked out according to the DNA sequence of lvgA by ourselves.The lvgA gene was amplified from the total cell DNA of L.pneumophila by PCR,and then inserted it into the coloning vector pMD18-T.The sequence of inserted fragment was sequenced,and the lvgA sequences from different L.pneumophila species have been compared through cltust W software.The change of lvgA gene transcription levels of L.pneumophila after incubation with THP-1.1 cells was detecred by real-time fluorescence quantitative RT-PCR.Real-time quantitative RT-PCR assay based on lvgA gene for quick detection of L.pneumophila in peripheral blood of people who was infected by L.pneumophila.The prediction result suggested that lvgA located at the outside of outermembrane.The similarities of nucleotide and amino acid sequences of lvgA genes from different L.pneumophila species were 96%~99% and 98%~99%,respectively.The transcription level of lvgA gene was remarkably up-regulated after infecting the host cells.The detection result of real-time fluorescent quantitative RT-PCR were positive for all the specimens of peripheral blood collected from the L.pneumophila infected people.The lvgA gene conserved presented in different species of L.pneumophila.The alteration of lvgA gene transcription level suggests that it is an important virulence factor for L.pneumophila.The real-time fluorescent quantitative RT-PCR has been established for detecting L.pneumophila,and the advantages including quickness,stability,sensitivity and specificity,which indicats that this method can be used for clinical laboratory diagnosis of L.pneumophila infection.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2010年第6期546-550,共5页
Chinese Journal of Zoonoses
基金
金华市科技计划项目(2009-3-053)资助
