猪链球菌4型GalR蛋白的生物信息学分析及原核表达

doi:10.16431/j.cnki.1671-7236.2021.02.005

摘要/Abstract

摘要： 试验旨在了解猪链球菌4型（Streptococcus suis serotype 4，SS4）转录调控因子GalR蛋白生物学信息并对其进行原核表达。使用相关生物信息学分析网站对SS4 SH1510菌株GalR蛋白进行序列同源性和功能结构域检索，并对信号肽、跨膜区和等电点进行预测；同时，对SS4 SH1510菌株GalR基因进行扩增、原核表达、纯化，并用SDS-PAGE分析重组蛋白的可溶性，用Western blotting鉴定重组蛋白的反应原性。氨基酸序列比对显示，SH1510菌株GalR蛋白与变形链球菌、无乳链球菌、绿脓杆菌、马链球菌兽疫亚种、肺炎链球菌、口腔链球菌及李斯特菌的GalR家族蛋白的氨基酸序列分别具有57%、56%、55%、55%、52%、52%和49%的同源性；结构域检索发现GalR蛋白N-末端具有螺旋-转角-螺旋（helix-turn-helix，HTH）基序的小DNA结合结构域，C-末端具有Ⅰ型周质结合蛋白折叠的调节配体结合域，在这两个功能结构域的中间含有一个约18-氨基酸的连接子；GalR蛋白无信号肽，无跨膜区，等电点为5.10。SDS-PAGE分析显示，GalR蛋白绝大部分表达在上清，分子质量为56 ku；Western blotting鉴定结果显示，His单克隆抗体和粗制SS4多克隆抗体兔血清能特异性识别可溶性GalR蛋白。本研究对GalR蛋白进行了生物信息学分析并成功地表达和纯化了GalR蛋白，为进一步研究GalR蛋白在SS4中的作用奠定了基础。

关键词: 猪链球菌4型(SS4); 转录调控因子GalR; 生物信息学; 原核表达

Abstract: The aim of this study was to understand the bioinformatics information of GalR protein,a transcription regulator of Streptococcus suis serotype 4 (SS4),and to express it in prokaryotic cells.The sequence homology and functional domain of GalR protein of SS4 SH1510 strain were searched by bioinformatics analysis website,and the signal peptide,transmembrane domain and isoelectric point were also predicted.At the same time,the GalR gene of SS4 SH1510 strain was amplified,prokaryotic expressed and purified.SDS-PAGE was used to analyze the solubility of the recombinant protein,Western blotting was used to identify the reactivity of the recombinant protein.The amino acid sequence alignment showed that it had 57%,56%,55%,55%,52%,52% and 49% homology with the GalR family proteins of Streptococcus mutans,Streptococcus agalactiae,Pseudomonas aeruginosa,Streptococcus equi subsp.zooepidemicus,Streptococcus pneumoniae, Streptococcus oralis and Listeria,respectively.Domain search showed that the GalR protein had a small DNA binding domain with a helix-turn-helix (HTH) motif at the N-terminus,and a ligand-regulated binding of type Ⅰ periplasmic binding protein folding at the C-terminus.In the middle of these two functional domains,there was a linker of about 18 amino acids.GalR protein had no signal peptide and transmembrane region,and its isoelectric point was 5.10.SDS-PAGE analysis showed that GalR protein was mostly expressed in the supernatant and the molecular weight was 56 ku.Western blotting identification showed that His monoclonal antibody and crude SS4 polyclonal rabbit serum could specifically recognize soluble GalR protein.A comprehensive bioinformatics analysis of the GalR gene was performed and the GalR protein was successfully expressed and purified in this study,which laid a foundation for further study of the role of GalR protein in SS4.

Key words: Streptococcus suis serotype 4 (SS4); transcriptional regulator GalR; bioinformatics; prokaryotic expression

中图分类号:

S852.61

孙珂, 祝昊丹, 周俊明, 王丹丹, 俞正玉, 吕立新, 何孔旺, 李彬, 倪艳秀. 猪链球菌4型GalR蛋白的生物信息学分析及原核表达[J]. 中国畜牧兽医, 2021, 48(2): 443-449.

SUN Ke, ZHU Haodan, ZHOU Junming, WANG Dandan, YU Zhengyu, LYU Lixin, HE Kongwang, LI Bin, NI Yanxiu. Bioinformatics Analysis and Prokaryotic Expression of GalR Protein of Streptococcus suis Serotype 4[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 443-449.

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