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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 PCR-based mce gene-based PCR assay to analyze the conserved mce gene of Leptospira interrogans (Leptospira interrogans) and to obtain bioinformatics analysis results of the mce gene from Leptospira interrogans. Methods The NCBI / Blast and NCBI / Cds softwares were used to search the conserved functional domains and the specificity of mce gene in Mce. The TMHMM Server-2.0 was used to predict the transmembrane domain structure of the encoded protein. Based on the reported mce gene Primers were designed and used to amplify the full-length mce gene of Lysimachiae pallidum by sequencing. DNA sequencing was performed using DNAStar software. Genomic DNA from 13 strains of Leptospira interrogans was used as a template to verify the mce gene PCR-based method. The amplified product is sequenced and aligned, and a phylogenetic tree is made based on the putative amino acid sequence. Results Bioinformatics studies showed that Mce contained a conserved functional domain of Mce superfamily associated with pathogenic bacteria adhesion and invasion. A clear transmembrane structure was found in Mce. At the same time, mce gene was specifically found in the tick. The cloned mce gene Which was consistent with the reported sequence (GenBank accession No.:NP_712236). The results of PCR showed that the mce genes were inherited by all the 13 M. constella strains in our country. The nucleotide and amino acid sequence identities were all above 95%. CONCLUSION: The mce gene of Leptospira interrogans is specific and conserved in all pathogenicity ticks. Mce has a distinct transmembrane structure and contains Mce superfamily domains, which are involved in the adhesion and invasion of host cells.