小反刍兽疫病毒H基因在杆状病毒中的表达及其免疫原性研究

Expression of H Gene of Peste des Petits Ruminants Virus in Baculovirus and Its Immunogenicity Study

为表达小反刍兽疫病毒(PPRV)H蛋白,并探讨其免疫原性,通过RT-PCR克隆PPRV Nigeria75/1毒株H基因,将其克隆至供体质粒pFB-LIC-Bse中,测序验证后将重组质粒pFB-LIC-Bse-PPRV-H转化至DH10Bac感受态细胞中,经同源重组获得重组杆状病毒穿梭质粒bacmid-PPRV-H,将其转染昆虫细胞sf21获得含有PPRV H基因的重组杆状病毒。采用SDS-PAGE、Western blot、IFA及小鼠免疫试验鉴定表达产物的反应原性及免疫原性。结果表明,在杆状病毒表达系统中表达的PPRV H蛋白能与His-tag单克隆抗体及PPRV阳性血清发生特异性反应,其相对分子质量为68ku;表达产物接种小鼠可诱导其产生特异性抗体和抗小反刍兽疫病毒中和抗体,淋巴细胞增殖试验检测结果表明重组杆状病毒rBacmid-H能与相应抗体和致敏的效应淋巴细胞发生较强的特异性反应。本试验获得的重组杆状病毒表达的PPRV H蛋白具有良好的免疫原性及反应原性,可诱导小鼠产生保护性中和抗体,该试验为进一步开发小反刍兽疫亚单位疫苗奠定了基础。

The aim of this study was to study the expression and immunogenicity of H protein of peste des petits ruminants virus(PPRV).The Hgene of PPRV Nigeria75/1strain was amplified by RT-PCR,and cloned into the donor plasmid pFB-LIC-Bse.After being sequenced,the recombinant plasmid pFB-LIC-Bse-PPRV-H was transformed into competent cells of E.coli DH10Bac to get recombinant shuttle plasmid.The recombinant shuttle plasmid was then transfected into sf21cells to get recombinant baculovirus.Western blot,IFA and immunity tests in mice were performed to identify the immunoreactivity and immunogenicity of the expression products.The recombinant H protein with molecular mass of approximately 68kDa was detected with His tag monoclonal antibody and PPRV positive serum.The exprsssion products possessed a favorable immunogenicity and fall immunized mice could produce anti-PPRV neutralizing antibody.The cellimmune responses were examined by lymphocyte proliferative assay using MTS method.The results showed that the rBacmid-H was able to express H proteins in Vero cells.Furthermore,specific antibodies were induced in the second week after primary vaccination.In conclusion,the recombinant H protein had good immunogenicity and immunoreactivity,which laid a foundation of developing a new vaccine against PPR.