摘要
对四川猪链球菌2型(S.suis 2)强毒株05ZYH33溶血素样蛋白(hemolysin-like protein,HLP)编码基因hlp进行序列信息分析、分子克隆表达及溶血活性检测,深入探讨HLP的致病机制.用Blast和Clustal X程序对HLP进行基因同源性分析,用Signal P 4.1和TMHMM Server 2.0分析HLP氨基酸序列.设计合成hlp引物进行PCR扩增,先后将hlp克隆入p MD-18T载体和p ET30a表达载体中,构建p ET30a::hly原核表达质粒.将重组质粒转化至E.coli BL21中诱导表达,并对重组HLP蛋白进行纯化和溶血活性检测.同源性分析表明HLP与多种具有溶血活性的蛋白相似性较高.氨基酸序列分析发现HLP具有信号肽和跨膜区,且由DUF21、CBS和Cor CHly C 3部分组成.序列测定显示hly片段全长1 335 bp,编码445个氨基酸.重组质粒经诱导表达和纯化后电泳显示,HLP分子量约为70 k Da,具有溶血活性.结果表明,HLP是一个膜结合蛋白,不同于能够分泌到细胞外的溶血素Suilysin.重组HLP具有一定的溶血效价,此可能与致病相关.
The paper analyzes the HLP sequence information, molecular cloning and detects the hemolytic activity of hemolysin-like protein（HLP） in Streptococcus suis serotype 2（ S. suis 2） in Sichuan province, in order to provide references for the study of the pathogenic mechanism. Blast and Clustal X are used to ana- lyze the homology of HLP, while SignalP 4.1 and TMHMM 2.0 are used for the analysis of HLP amino acid sequence. ~ primers are designed and synthesized to amplify the PCR method, and then HIP is cloned into the pMD-18T vector, and then cloned into the expression vector pET30a to construct pET30a： ： hly prokaryotic expression plasmid. The recombinant expression plasmid is transformed into E. coli BL21 to in- duce expression, and the expression of the protein is purified, and then the hemolytic activity of HIP is de- tected. Homology analysis shows a highly homologous of HLP with other hemolytic proteins. Amino acid se- quence analysis finds that HIP has both signal and transmembrane segments, and is made up of DUF21, CBS and CorC-HlyC. Sequence determination shows the length of hly fragment is 1 335 bp, and 445 amino acids are coded. After the recombinant plasmid is induced to express and purified, a 70 kDa protein with hemolytic activity could be observed. The conclusion drawn from the paper is that HIP is a membrane- bound protein, unlike Suilysin that can be secreted into the extraeellular. Recombinant HIP has a certain hemolytic titer, which may be related to pathogenesis.
出处
《成都大学学报（自然科学版）》
2016年第3期209-214,共6页
Journal of Chengdu University（Natural Science Edition）
基金
国家自然科学基金(31300119)资助项目
