B族链球菌C5a肽酶表位的预测、分段表达及其免疫原性

Epitope Prediction,Fractional Expression and Immunogenicity of Streptococcal C5a Peptidase from Group B Streptococcus

目的应用生物信息学方法预测B族链球菌C5a肽酶蛋白(SCPB)的表位,分段表达其中4个功能活性区域,并分析其免疫原性。方法用预测程序ProPred和ANTIGENIC预测SCPB的表位;分别构建C5a肽酶4个目的片段的重组表达质粒,经酶切测序正确后,转化E. coli BL21(DE3),IPTG诱导表达,SDS-PAGE分析目的蛋白的表达形式及表达量;并经镍柱亲和层析纯化,纯化的重组蛋白进行蛋白质谱分析和Western blot分析后,皮下免疫小鼠,ELISA检测小鼠血清抗体水平。结果经预测,SCPB含1个既可结合MHC又具有B细胞表位特征的肽段。构建的4个重组表达质粒经酶切及测序鉴定正确,目的蛋白主要以可溶性形式表达,表达量约占菌体总蛋白的30%～70%。纯化的重组蛋白纯度可达90%,质谱分析表明与SCPB的相似性很高;Western blot分析表明,可与兔抗全长SCPB多克隆抗体反应。重组F1、FE、Fn蛋白免疫小鼠血清抗体滴度较高,三者差异无统计学意义;F2a蛋白抗体滴度最低,与F1、FE和Fn蛋白差异有统计学意义。结论已成功构建并高效表达了SCPB的4个功能活性区域;Fn是重要的免疫优势表位功能区;本文为B族链球菌毒力机制的研究及亚单位蛋白疫苗的研制奠定了基础。

Objective To predict the epitopes,fractionally express the four functional domains and analyze the immunogenicity of streptococcal C5a peptidase from group B streptococcus（SCPB）.Methods The epitopes of SCPB were predicted by using ProPred and ANTIGENIC softwares.The recombinant plasmids carrying the four target gene fragments of SCPB were constructed respectively,identified by restriction analysis and sequencing,then transformed to E.coli BL21（DE3）for expression under induction of IPTG.The target protein was analyzed for form and expression level by SDS-PAGE,and purified by nickel ion affinity chromatography,then analyzed by peptide mass fingerprinting（PMF）and Western blot.C57 /BL mice were immunized s.c.with the purified protein and determined for serum antibody.Results It was predicted that SCPB contained a peptide fragment which could bind to MHC and showed the characters of B cell epitope.Both restriction analysis and sequencing proved that the four recombinant plasmids were constructed correctly.The expressed products mainly existed in soluble forms and contained about 30%~70% of total somatic protein.The purified recombinant proteins reached purities of 90%,and showed high homologies to SCPB as proved by PMF.Western blot showed specific reaction of the purified recombinant protein with rabbit polyclonal antibody against full-length of SCPB.High antibody titers were induced in the sera of mice by recombinant F1,FE and Fn proteins,which showed no significant difference.However,the antibody titer induced by F2a protein was significantly lower than those by the above-mentioned three kinds of proteins.Conclusion The four functional domains of SCPB were successfully constructed and highly expressed,and Fn was an important functional domain of prominent immune epitope,which laid a foundation of study on virulence mechanism of group B streptococcus（GBS）and development of GBS subunit vaccine.