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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 of C5a peptidase protein (SCPB) of Group B Streptococcus pneumoniae by bioinformatics methods and express four functional active regions in segments and analyze its immunogenicity. Methods The predicted epitopes of SCPB were predicted by ProPred and ANTIGENIC. The four recombinant plasmids were constructed and sequenced. The recombinant plasmids were transformed into E. coli BL21 (DE3) and induced by IPTG. SDS-PAGE The expression and expression of the target protein were analyzed. The recombinant protein purified by Ni-P affinity column was analyzed by protein profile and Western blot, then the mice were immunized subcutaneously and the level of serum antibody was detected by ELISA. Results It is predicted that SCPB contains one peptide that binds both MHC and B-cell epitopes. The constructed recombinant plasmids were identified by restriction enzyme digestion and sequencing. The target protein was mainly expressed in soluble form, which accounted for about 30% -70% of the total bacterial proteins. Purified recombinant protein purity of up to 90%, mass spectrometry showed high similarity with SCPB; Western blot analysis showed that the rabbit anti-full-length SCPB polyclonal antibody reaction. The antibody titers of F1, FE and Fn immunized mice were higher, but there was no significant difference among the three groups. The antibody titer of F2a protein was the lowest, and the difference was statistically significant between F1, FE and Fn protein. Conclusions Four functional active regions of SCPB have been successfully constructed and expressed. Fn is an important immunodominant epitope functional region. This study lays the foundation for the study of virulence mechanism of Streptococcus group B and the development of subunit protein vaccine.