PRRSV中和表位串联Fc分子的表达与免疫原性鉴定

Expression and immunogenicity analysis of Fc fragment in tandem with neutralizing epitopes of porcine reproductive and respiratory syndrome virus

为融合表达猪繁殖与呼吸综合征病毒(PRRSV)中和表位和Fc分子,首先合成PRRSVGP3和GP5的串联B细胞中和表位与鼠IgG1-Fc的mFc-GP35-Bp基因片段,将其克隆到pFastBac载体上,构建重组质粒pFastBac-mFc-GP35-Bp,经蓝白斑筛选出重组杆粒Bacmid-mFc-GP35-Bp,并转染昆虫Sf9细胞,获得重组杆状病毒rBV-mFc-GP35-Bp。通过Western-blot分析重组蛋白的反应原性,免疫小鼠后,通过间接ELISA检验其免疫原性,病毒中和试验检测中和抗体水平。结果显示,mFc-GP35-Bp蛋白在昆虫Sf9细胞中获得表达,且可分泌至上清;用mFc-GP35-Bp蛋白免疫小鼠后,ELISA测定抗体效价可达1∶100000,三免后第4周仍可检测到较高抗体水平;中和抗体效价介于1∶40和1∶80之间。结果表明,表达的重组mFc-GP35-Bp蛋白具有良好的反应原性和免疫原性,可刺激机体产生中和抗体,与Fc分子融合表达延长了抗体在血清中存在的时间,本试验为PRRSV中和表位疫苗的研发奠定了基础。

To fuse the neutralizing epitopes and of the porcine reproductive and respiratory syndrome virus(PRRSV)and Fc molecule,the mFc-GP35-Bp gene fragment consisting of concatenated B cell neutralizing epitopes of PRRSV GP3 and GP5 and mouse IgG1-Fc was synthesized and cloned into the pFastBac vector.The recombinant plasmid pFastBac-mFc-GP35-Bp was constructed,and the recombinant baculovirus rBV-mFc-GP35-Bp was obtained by transfecting the insect Sf9 cells after blue-white screening of the recombinant bacmid Bacmid-mFc-GP35-Bp.The reactivity of the recombinant protein was confirmed by Western-blot analysis,and immunogenicity was evaluated by immunizing mice and detecting antibody titers by indirect ELISA and virus neutralization tests.The results showed that the mFc-GP35-Bp protein was expressed in the insect Sf9 cells and could be secreted into the supernatant.Immunization with the mFc-GP35-Bp protein produced antibodies with titers up to 1∶100000,which remained high even in the fourth week after the third immunization.The neutralizing antibody titer was between 1∶40 and 1∶80.These findings indicate that the expressed recombinant mFc-GP35-Bp protein has good reactivity and immunogenicity,stimulates the production of neutralizing antibodies,and extends the duration of antibodies in the serum through fusion with the Fc molecule.This study provides a foundation for the development of PRRSV neutralizing epitope vaccines.