摘要
【目的】单增李斯特菌是一种重要的食源性致病菌,常引起人和动物致病。其细胞壁表面LPXTG基序蛋白在单增李斯特菌致病过程中发挥重要作用,根据参考株全序列预测的41个LPXTG基序蛋白中仍有部分蛋白功能未知。对单增李斯特菌新疆绵羊脑分离株LM90SB2的LPXTG基序蛋白Lmo0160的基因进行克隆及生物信息学分析,为功能验证提供基础。【方法】根据GenBank中收录的lmo0160序列设计特异性引物,利用PCR方法对新疆分离株的lmo0160基因进行扩增,将扩增产物克隆到pMD 19-T载体,进行PCR、双酶切鉴定及序列测定,并对基因核苷酸序列和蛋白序列进行分析。【结果】分离株LM90SB2的lmo0160序列全长为1 708 bp,包含1 428 bp的开放阅读框,共编码475个氨基酸;LM90SB2株lmo0160核苷酸序列与CFSAN008100株(4b型,美国)、CFSAN023463株(4b型,美国)、J2-064株(4b型,美国)、F2365株(4b型,奶酪,美国)和NTSN株(4b型,绵羊脑,中国扬州)相似性为99.0%-99.1%;与M7株(4a型,牛奶,中国浙江)相似性为97.2%,与Finland1998株(1/2a型,美国)、N53-1株(1/2a型,熟火腿,瑞士)、N1546株(1/2a型,鱼,丹麦)和EGD-e株(1/2a型,美国)相似性为87.2%-91.1%;其推导的氨基酸序列与上述菌株相似性为91.8%-99.4%。系统进化树显示,LM90SB2菌株的lmo0160基因与CFSAN023463、F2365和NSTN菌株亲缘关系较近,处于同一分支上,而与标准株EGD-e菌株亲缘关系较远。蛋白质二级结构预测表明,LM90SB2的Lmo0160蛋白为亲水性蛋白,无信号肽,不形成跨膜结构。蛋白结构域预测表明Lmo0160蛋白含有胶原蛋白结合域和Cna B结构域。【结论】克隆了LM90SB2的lmo0160基因,为进一步研究LM90SB2的lmo0160基因功能奠定了基础。
[Objective] Listeria monocytogenes, an important food-borne pathogenic bacteria, often causes human and animal diseases. The cell wall surface proteins with an LPXTG motif play an important role in the pathogenic process of L. monocytogenes. According to the whole sequence of the reference strain, the function of some proteins among the 41 predicted LPXTG motif proteins is unknown. The aim of this study is to clone and bioinformatics analysis of the gene of LPXTG motif protein Lmo0160 of L. monocytogenes LM90SB2 isolated from diseased sheep in Xinjiang. It could provide the foundation for functional verification. [Methods] A pair of specific primers was designed according to the predicted sequence of L. monocytogenes lmo0160 gene in GenBank. The lmo0160 gene was amplified by PCR, and the amplified products were cloned into p MD19-T vector and verified by PCR, restriction endonuclease digestion and sequencing. In addition, the bioinformatics on nucleotide sequence of lmo0160 gene of LM90SB2 strain as well as putative protein structure had been analyzed. [Results] The sequence length of LM90SB2 lmo0160 gene was 1708 bp, containing a 1 428 bp open reading frame（ORF） encoding 475 amino acids. The nucleotide sequence similarity of lmo0160 gene of LM90SB2 strain was 99.0%-99.1% with FSAN008100 strain（serotype 4 b, USA）, CFSAN023463 strain（serotype 4 b, USA）, J2-064 strain（serotype 4 b, USA）, F2365 strain（serotype 4 b, cheese isolate, USA） and NTSN strain（serotype 4 b, sheep brain isolate, Yangzhou, China）, 97.2% with M7 strain（serotype 4 a, cow＇s milk isolated, Zhejiang, China）, and 87.2%-91.1% with Finland 1998 strain（serotype 1/2 a, USA）, N53-1 strain（serotype 1/2 a, fish isolate, Denmark）, N1546 strain（serotype 1/2 a, cooked ham isolate, Switzerland） and EGD-e strain（serotype 1/2 a, USA）. Its putative amino acid sequence similaritywas 91.8%-99.4% with above strains. The phylogenetic tree showed that the lmo0160 gene of LM90SB2 strain was closely relative with CFSAN023463
出处
《微生物学通报》
CAS
CSCD
北大核心
2017年第12期2905-2913,共9页
Microbiology China
基金
国家自然科学基金项目(No.31360614)~~
