问号钩端螺旋体mce基因及其编码蛋白特征的初步分析

Preliminary analysis of the characteristics of Leptospira interrogans mce gene and its encoded protein

目的建立基于mce基因的PCR检测方法,分析问号钩端螺旋体(钩体)mce基因保守性,获得钩体mce基因生物信息学分析结果。方法以NCBI/Blast、NCBI/Cds软件搜索Mce内部存在的保守功能结构域及mce基因的特异性;采用TMHMM Server-2.0预测所编码蛋白跨膜区结构;根据已报道问号钩体赖株mce基因设计引物,采用PCR扩增问号钩体赖株全长mce基因,测序并使用DNAStar软件进行测序结果比对。以13株国内流行问号钩体菌株基因组DNA为模板,对基于mce基因PCR检测方法进行验证。扩增产物进行测序、比对,同时根据推定的氨基酸序列做系统发生树。结果生物信息学研究表明,Mce含有与病原菌黏附侵袭相关的Mce超家族保守功能结构域;Mce中存在一明显的跨膜结构;同时,mce基因特异存在于问号钩体中;所克隆的mce基因与已报道序列(GenBank accession No.:NP_712236)一致;PCR检测结果表明,我国13株问号钩体株均携带mce基因;核苷酸和氨基酸序列相似性均>95%。结论问号钩体mce基因特异、保守存在于各致病性的问号钩体中;Mce具有明显的跨膜结构并含有Mce超家族结构域,与问号钩体黏附侵袭宿主细胞有关。

Objective To establish a mce gene-based PCR detection method for the analysis of mce gene conservation of Leptospira interrogans and its bioinformatics.Methods The NCBI/Blast and NCBI/Cds online database was used for the search of the conserved functional domain in the mce gene and its specificity,with TMHMM Server-2.0 employed for the prediction of the transmernbrane regions of the encoded protein.The segments of entire mce gene from L.interrogans strain lai were amplified by PCR and then sequenced,with the sequence of cloned mce gene compared with the reported sequence of mce gene（GenBank accession No.： NP_712236） by DNAStar.The genome DNAs from the 13 prevalent strains of L.interrogans in China were used as the templates for the validation of the mce gene-based PCR detection method.A comparative analysis of the amplified mce gene products from different strains was also accomplished based on their sequences and a phylogenetic tree was generated from the presumed sequences of amino acids.Results The results of bioinformatic analysis showed that there was a mce super family conservation functional domain that was associated with adhesion and invasion of pathogens in the mce gene from L.interrogans.It was clear that there was a L.interrogans transmembrane structure in mce.High homology sequences of mce gene were present in L.interrogans,with the similarities of nucleotide sequences of mce gene from different L.interrogans strains more than 95%.Conclusion The mce gene with conservation specifically exists in different pathogenic L.interrogans strains,and it has a transmembrane structure and a super family functional domain that is associated with L.interrogans adhering to and invading their host cells.