牛细小病毒病毒样颗粒的组装及其免疫原性评价

Assembly and immunogenicity of bovine parvovirus virus-like particles

为研究牛细小病毒(BPV)VP2衣壳蛋白自组装病毒样颗粒(VLPs)以及VLPs的免疫原性。本研究以BPV-1型C7-5中国分离株的基因组序列为模板扩增VP2蛋白的编码基因,将其克隆至腺病毒穿梭载体p Shuttle-CMV中,利用E.coli BJ5183内同源重组将VP2基因插入腺病毒骨架质粒p Ad Easy-1中,获得携带BPV VP2基因的重组腺病毒质粒(p Ad-BPV-VP2)。该重组质粒经PacⅠ线性化后转染Ad293细胞,获得重组腺病毒r Ad-BPV-VP2,第8代时滴度为107.25TCID50/m L。间接免疫荧光、western blot以及电镜观察结果显示,BPV VP2蛋白可以在腺病毒系统中稳定表达,并有效组装VLPs。将r Ad-BPV-VP2肌肉注射免疫BALB/c小鼠,结果显示,针对BPV的血清Ig G和中和(VN)抗体分别于加强免疫后2周和8周达最高水平,高峰抗体可持续至15周,VN抗体的最高滴度可达1∶4 000。此外,与对照小鼠血清相比,免疫小鼠血清中的IL-2、IL-4及IFN-γ含量显著升高(p<0.05)。结果表明,r Ad-BPV-VP2免疫小鼠后可以诱导机体产生针对BPV的特异性体液和细胞免疫。本研究为BPV的免疫预防及载体的开发奠定了基础。

To evaluate the efficiency of self-assembling the VP2 capsid proteins of bovine parvovirus （BPV） to virus-like particles （VLPs） and the immunogenicity of the VLPs,the BPV VP2 gene was amplified using the genomic DNA of BPV-1 Chinese isolate C7-5 as a template and cloned into the pShuttle-CMV plasmid.Recombinant adenovirus plasmid pAd-BPV-VP2 was constructed by homologous recombination with the adenoviral backbone plasmid pAdEasy-1 in E.coli BJ5183 cells,which was linearized by Pac Ⅰ and transfected into Ad293 cells for package to generate the recombinant adenovirus rAd-BPV-VP2.The titer of the rAd-BPV-VP2 reached to 107.25 TCID50/mL after eighth passages in Ad293 cells,and the VP2 protein of BPV was stably expressed and assembled efficiently into VLPs in rAd-BPV-VP2 infected cells detected by indirect immunoflourescent assay,western blot and electronic microscopy.In addition,anti-BPV IgG and viral neutralizing （VN） antibody were induced in mice by intramuscular inoculation with rAd-BPV-VP2.The peak titers of the serum IgG and VN antibody （1：4 000） against BPV were produced at 2 and 8 weeks post-inoculation （p.i.）,respectively,and the durations of the highest titers of IgG and VN antibody were lasted for 15 weeks.Furthermore,comparing to the control serum,the level of IL-2,IL-4 and IFN-γ increased significantly （p＜0.05） in the serum of rAd-BPV-VP2 immunized group.These results demonstrated that the rAd-BPV-VP2 was able to induce high levels of both humoral and cellular immunoresponses against BPV infection.