罗非鱼无乳链球菌C5a肽酶的克隆及原核表达质粒构建

Cloning and construction of recombinant expression plasmid of C5a peptidase of Streptococcus agalactiae isolated from tilapia

为进一步了解无乳链球菌表面蛋白C5a肽酶scpB基因免疫表位及毒力作用基团的相关信息,通过提取罗非鱼无乳链球菌基因组DNA,利用特异性引物PCR扩增出scpB基因的全长开放阅读框(ORF)。结果表明,该基因包括3 405 bp的ORF,可编码1 134个氨基酸,与已报道的人源无乳链球菌ScpB的氨基酸同源性达99.74%。利用生物信息学软件DNAstar、Clustal X 2.0和MEGA 5.05及在线分析工具ExPASy ProtParam、NCBIProten blast及Conserved Domain等对推导的氨基酸序列进行分析,结果显示罗非鱼无乳链球菌scpB基因编码的氨基酸序列含有3个催化三联体(catalytic triad)位点、7个假定活性位点(putative active site)、2个特异位点(spe-cific hits),以及4个超家族结构(2个肽酶超家族结构Peptidases-S8-S53 superfamily、1个类纤连蛋白超家族结构DUF1034 superfamily和1个鞭毛钩蛋白超家族结构FlgD-lg superfamily);并且该氨基酸序列有多个抗原表位,推测ScpB蛋白应具有较强的免疫原性,可作为候选的蛋白疫苗成分。将该片段插入原核表达载体pET32a(+),转入大肠杆菌BL21(DE3),挑取阳性克隆,经PCR、酶切及测序鉴定表明成功构建原核表达载体pET32a(+)/scpB。

Streptococcus agalactiae surface proteins can stimulate the host to produce protective immune.The protein C5a peptidase is widespread in a variety of serotypes of Streptococcus strains,highly conserved and has good immunogenicity.It is a possible carrier protein that could induce protective immune response by itself.However,the immune epitopes and the role of virulence groups of S.agalactiae scpB gene of fish is still unclear.In this study,the scpB gene was amplified from genome DNA of S.agalactiae isolated from tilapia by PCR with specific primers.Restriction analysis and DNA sequencing confirmed that the scpB gene has a ORF of 3 405 bp,encoding 1 134 amino acid,which was highly conserved and had a surprising degree of homology among strains isolated from other mammals.Molecular analysis of scpB gene was performed by bioinformatics tools such as DNAstar,Clustal X 2.0,MEGA 5.05,ExPASy ProtParam,NCBI Protein blast,and NCBI Conserved Domain et al.The results showed that the amino acid encoded by the scpB gene contained 3 catalytic triad,7 putative active sites,2 specific hits,and 4 super families（2 Peptidases-S8-S53,1 DUF1034,and 1 FlgD-lg）.Moreover,it was found that C5a peptidase had mutiple epitopes,suggesting that the protein may have strong immunogenicity.Then the PCR product was inserted into pET32a（＋） and the constructed recombinant plasmid pET32a（＋）/scpB was transformed to E.coli BL21（DE3） for expression.The positive colony,which were identified by PCR and digestion（EcoRⅠand XhoⅠ）,showed that the recombinant plasmid pET32a（＋）/scpB was constructed successfully.This study paved the way for further study of immune mechanism and polypeptide vaccine based on C5a peptidase from the tilapia S.agalactiae.