单增李斯特菌llsB基因克隆及序列分析

doi:10.16431/j.cnki.1671-7236.2019.07.002

摘要/Abstract

摘要：

试验旨在对新疆绵羊脑炎临床分离株LM90SB2单增李斯特菌llsB基因进行克隆及生物信息学分析，以期进一步完善单增李斯特菌溶血素S的功能研究。根据GenBank中单增李斯特菌F2365基因全长序列（登录号为：AE017262）设计其特异性引物，利用PCR方法对新疆分离株LM90SB2的llsB基因进行扩增，回收目的基因与pMD19-T载体连接，采用PCR、双酶切鉴定筛选阳性菌并进行测序，对所获序列进行同源性比对及遗传变异分析。结果显示，新疆分离株LM90SB2的llsB基因序列全长为876 bp，共编码291个氨基酸；LM90SB2分离株llsB基因核苷酸序列与10-0809、81-0592、81-0558、02-1792、NTSN、02-1289不同分离株同源性均为100%，与CⅡMS-PH-1、NRRLB-57603株的同源性均为99.9%，与J1816、R2-502株的同源性为44.7%~45.0%。分子进化树显示，LM90SB2菌株llsB基因与血清型为4b的菌株亲缘关系较近，聚类为同一分支。蛋白质二级结构预测表明，LM90SB2 llsB蛋白为亲水性蛋白，无信号肽，不形成跨膜结构。本试验成功克隆了LM90SB2株llsB基因，为深入探讨该基因功能提供全面的理论依据。

关键词: 单增李斯特菌; llsB基因; 克隆; 生物信息学分析

Abstract:

The aim of this study was to clone and analyze llsB gene of Listeria monocytogenes LM90SB2 isolated from encephalitis sick sheep in Xinjiang,and further improve the functional study of listeriolysion S.Specific primers were designed according to the full-length sequence of Listeria monocytogenes F2365 gene in GenBank (accession No.:AE017262),llsB gene of Xinjiang isolate LM90SB2 was amplified by PCR,and recycled the target gene and connected with pMD19-T vector.Positive bacteria was identified and screened for sequencing by PCR and double enzyme digestion methods.The nucleotide sequence with the correct sequencing result was analyzed,and the homology and genetic variation analysis were carried out.The results showed that llsB gene of Xinjiang isolate LM90SB2 was 876 bp in length and encoded a total of 291 amino acids.The nucleotide homology of llsB gene were 100% between LM90SB2 isolate and 10-0809,81-0592,81-0558,02-1792,NTSN and 02-1289 isolate strains,respectively,the homology with CⅡMS-PH-1 and NRRLB-57603 were both 99.9%,and the homology with J1816 and R2-502 were 44.7% to 45.0%.The molecular phylogenetic tree showed that llsB gene of LM90SB2 strain was closely related to the strain with serotype 4b,and the clustering was the same branch.Protein secondary structure prediction results showed that LM90SB2 llsB protein was a hydrophilic protein,without signal peptide and transmembrane structure.This experiment successfully cloned LM90SB2 llsB gene,and provided a comprehensive theoretical basis for the function research of LM90SB2 llsB gene.

Key words: Listeria monocytogenes; llsB gene; cloning; bioinformatics analysis

中图分类号:

S852.61

王欣雨, 尹华, 任静静, 李红欢, 蒋建军. 单增李斯特菌llsB基因克隆及序列分析[J]. 《中国畜牧兽医》, 2019, 46(7): 1890-1898.

WANG Xinyu, YIN Hua, RENG Jingjing, LI Honghuan, JIANG Jianjun. Cloning and Sequence Analysis of Listeria monocytogenes llsB Gene[J]. , 2019, 46(7): 1890-1898.

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