首页 | 期刊介绍 | 编委会 | 道德声明 | 投稿指南 | 常用下载 | 过刊浏览 | 期刊订阅 | In English
引用本文:樊博琳,潘丽霞,王忠良,黎源,简纪常,王蓓.罗非鱼源无乳链球菌(Streptococcus agalactiae)新型AI-2信号分子受体RbsB蛋白结晶生长研究.海洋与湖沼,2020,51(1):132-140.
【打印本页】   【HTML】   【下载PDF全文】   查看/发表评论  下载PDF阅读器  关闭
←前一篇|后一篇→ 过刊浏览    高级检索
本文已被:浏览 79次   下载 95 本文二维码信息
码上扫一扫!
分享到: 微信 更多
罗非鱼源无乳链球菌(Streptococcus agalactiae)新型AI-2信号分子受体RbsB蛋白结晶生长研究
樊博琳1, 潘丽霞2, 王忠良1, 黎源1, 简纪常1,3,4, 王蓓1,3,4
1.广东海洋大学水产学院 广东省水产经济动物病原生物学及流行病学重点实验室 广东省水产经济动物病害控制重点实验室 湛江 524088;2.广西科学院 南宁 536000;3.中国科学院实验海洋生物学重点实验室 中国科学院海洋研究所 青岛 266071;4.海洋生物学与生物技术功能实验室 青岛海洋科学与技术试点国家实验室 青岛 266071
摘要:
为了开展无乳链球菌(Streptococcus agalactiae)核糖结合蛋白(Ribose binding protein B,RbsB)结构功能的研究,本实验根据已知无乳链球菌ZQ0910全基因组序列设计相关引物。采用PCR方法扩增其RbsB基因,随后将该基因定向克隆到原核表达载体pGEX-6p-1中,在大肠杆菌BL21(DE3)感受态细胞中进行IPTG诱导表达;采用HRV 3C蛋白酶切除GST标签,分子筛分离获得RbsB蛋白;运用生物信息学软件对RbsB基因序列进行分析,并对RbsB蛋白二级和三级结构进行预测;采用NeXtal Tubes JCSG Core Suite结晶试剂盒筛选蛋白结晶条件。研究结果表明,该基因全长为969碱基,编码322个氨基酸,RbsB蛋白理论分子量33.9ku,等电点为9.41,二级结构中α螺旋结构所占比重最高,建立RbsB蛋白三维结构模式图;经IPTG诱导后表达的融合蛋白分子量为59ku,筛选RbsB蛋白的初始结晶条件为(0.2mol/L di-Potassium hydrogen phosphate,20%(W/V)PEG3350;1.5mol/L ammonium sulfate,25%(V/V)Glycerol),获得RbsB蛋白结晶体。本研究结果可为无乳链球菌核糖结合蛋白(RbsB)的功能解析提供实验及理论基础。
关键词:  无乳链球菌  RbsB蛋白  蛋白纯化  结晶化
DOI:10.11693/hyhz20190500103
分类号:Q789
基金项目:国家自然科学基金项目,31702386号;广东省科技厅国际合作领域项目,2017A050501037号。
CRYSTAL GROWTH OF NOVEL AI-2 SIGNALING MOLECULE RECEPTOR RbsB PROTEIN FROM STREPTOCOCCUS AGALACTIAE
FAN Bo-Lin1, PAN Li-Xia2, WANG Zhong-Liang1, LI Yuan1, JIAN Ji-Chang1,3,4, WANG Bei1,3,4
1.Fisheries College of Guangdong Ocean University, Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemilogy for Aquatic Economic Animals, Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes, Zhanjiang 524088, China;2.Guangxi academy of Science, Nanning 536000, China;3.Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;4.Laboratory for Marine Biology and Biotechnology, National Laboratory for Marine Science and Technology, Qingdao 266071, China
Abstract:
To understand the structural function of the RbsB (Ribose binding protein B, RbsB) protein in Streptococcus agalactiae, we designed primers according to the related genes registered on GenBank, and amplified the RbsB gene of the strain by PCR (polymerase chain reaction). The gene was directionally cloned into the prokaryotic expression vector pGEX-6p-1. In addition, we carried out IPTG-induced expression in E. coli BL21 (DE3) competent cells, isolated a single RbsB protein by excising GST-tag with a kit (PierceTM HRV 3C Protease Solution Kit), analyzed the RbsB gene sequence with bioinformatics software, predicted the secondary tertiary structure of RbsB protein, and screened the protein crystallization conditions in the NeXtal Tubes JCSG Core Suite crystallization kit. The results show that the full-length of the gene was 969bp and 322aa, the theoretical molecular weight of RbsB protein was 33.9ku, and the isoelectric point was 9.41. The α-helix structure had the highest proportion in the secondary structure, and the three-dimensional structure pattern of RbsB protein was established; the molecular weight of the fusion protein was 59ku, the RbsB protein crystals could be obtained by initial crystallization conditions for screening (0.2mol/L di-Potassium hydrogen phosphate, 20% (W/V) PEG3350; 1.5mol/L ammonium sulfate, 25% (V/V) Glycerol). Thus, the RbsB protein crystals have been successfully obtained, which lays a foundation for the later study of the structure and function of RbsB protein.
Key words:  Streptococcus agalactiae  RbsB protein  protein purification  crystallization
版权所有 《海洋与湖沼》编辑部 Copyright©2008 All Rights Reserved
主管单位:中国科学技术协会 主办单位:中国海洋湖沼学会 中国科学院海洋研究所
地址:青岛市南海路七号  邮编:266071  电话:0532-82898753  E-mail:ols@qdio.ac.cn
技术支持:北京勤云科技发展有限公司