PCV2 ORF2 B细胞表位与PPV VP2真核表达质粒的构建及其免疫原性

为了研究猪圆环病毒2型(PCV2)ORF2B细胞表位与猪细小病毒(PPV)VP2重组真核表达质粒的免疫原性,将PCV2ORF2的B细胞表位基因(141～257bp)重组到PPV SC-1株VP2基因的N端,构建了真核表达质粒pCI-VP2.ORF2B。用脂质体转染法将重组质粒pCI-VP2.ORF2B转染至Vero细胞中,利用间接免疫荧光法检测其在体外的表达情况。将重组质粒免疫小鼠,并设猪细小病毒灭活疫苗、猪圆环病毒亚单位疫苗和空载体对照组,采用MTT比色法、流式细胞术和ELISA法分别对免疫小鼠脾淋巴细胞的转化功能,外周血CD4+和CD8+淋巴细胞比例,PCV2和PPV IgG抗体效价进行了检测。结果表明,转染Vero细胞后第48小时用间接免疫荧光法能在荧光显微镜下观察到绿色荧光,检测到特异性蛋白的表达;pCI-VP2.ORF2B免疫组脾淋巴细胞从第7天开始对ConA有明显反应,显著高于对照组;CD3+CD4+、CD3+CD8+T淋巴细胞的数量高于或显著高于对照组;在免疫后第14天检测到PPV/PCV2IgG抗体。提示pCI-VP2.ORF2B能够有效诱导机体产生细胞免疫和体液免疫反应。

Construction and immunogenicity of eukaryotic plasmid expressing B lymphocyte epitope gene of ORF2 of porcine circovirus type 2 fused with VP2 gene of porcine parvovirus

In order to investigate the immunogenicity of eukaryotic expression plasmid pCI-VP2.ORF2B,the B lymphocyte epitope gene of ORF2 of porcine circovirus type 2(PCV2) was recombined with the N terminus of VP2 gene of porcine parvovirus(PPV).Then the plasmid was transfected into Vero cells by liposome-mediated method and detected by indirect immunofluorescent assay.The recombinant plasmid pCI-VP2.ORF2B,the plasmid pCI-neo as control group,the inactivated PPV vaccine and PCV2 subunit vaccine were injected into mice by intramuscular route.The immune efficacy was detected by the MTT,flow cytometry and ELISA.It showed that the Vero cells with the transfection recombinant plasmid could be detected the specific expression protein at hour 48 post-inoculation(PI).The splenic lymphocytes had obvious response to ConA in the group of mice immunized by pCI-VP2.ORF2B on day 7 PI,which reactinogenicity was higher than the control group,and the number of CD3+CD4+and CD3+CD8+T lymphocytes was higher or significantly higher than the control group.The IgG antibody to PPV and PCV2 were detected on day 14 PI.These results manifested that the pCI-VP2.ORF2B induced effectively humoral and cellular immunity response to mice.