共表达猪IFN-γ基因对PPV VP2-PCV ORF2真核质粒的分子免疫佐剂效应研究  

Co-expression of Porcine INF-γ Gene and Its Molecular Adjuvant Effects on the PPV VP2-PCV2 ORF2 Eukaryotic Plasmid

作　　者：陈燕凌[1] 徐志文[1] 郭万柱[1] 朱玲[1] 徐凯[1] 唐玉香[1] 

机构地区：[1]四川农业大学动物生物技术中心动物疫病与人类健康实验室,雅安625014

出　　处：《畜牧兽医学报》2010年第10期1274-1280,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(30500019);四川省青年科技基金(2006Q14-049);四川省教育厅重点实验室项目(07ZZ027);"长江学者和创新团队发展计划"创新团队项目(IRT0848)

摘　　要：将干扰素-γ、猪圆环病毒Ⅱ型ORF2主要抗原表位(47-85位AA)和猪细小病毒VP2基因克隆至pCI-neo真核载体中,构建重组质粒pCI-IFN-γ.ORF2.VP2。用脂质体介导法将其转染到MDBK细胞中,利用RT-PCR和间接免疫荧光法检测产物的表达情况。将重组质粒pCI-IFN-γ.ORF2.VP2、pCI-ORF2.VP2、对照质粒pCI-neo、PBS、猪细小病毒灭活苗和猪圆环病毒亚单位疫苗通过肌肉注射免疫小鼠,检测小鼠不同时期的淋巴细胞转化功能、T淋巴细胞亚群动态变化情况以及PPV、PCV2抗体水平。试验结果显示,在转染重组质粒的MDBK细胞中能扩增到ORF2-VP2和IFN-γ基因;转染后48h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-IFN-γ.ORF2.VP2免疫组从第7天开始脾淋巴细胞对ConA有明显反应,显著高于对照组和PCI-ORF2.VP2免疫组;CD3+CD4+、CD3+CD8+T淋巴细胞的数量高于或显著高于对照组和pCI-ORF2.VP2免疫组;在免疫后第14天检测到PPV PCV抗体。结果表明,pCI-IFN-γ.ORF2.VP2能够有效诱导机体产生细胞免疫和体液免疫。Three genes encoding interferon-gamma（IFN-γ）,major epitope（47-85AA） of ORF2 of PCV2 and VP2 of Porcine parvovirus were cloned and inserted into eukaryotic vector of pCI-neo,the recombinant expression plasmid of pCI-IFN-γ.ORF2.VP2 was constructed.And then the constructed plasmid was transfected into MDBK cells by lipofectamine.The expressed product was tested by Indirect Immunofluorescence and RT-PCR.The recombinant plasmid pCI-IFN-γ.ORF2.VP2 and pCI-ORF2.VP2,the control plasmid pCI-neo,PBS,the PPV inactivated vaccine and PCV2 subunit vaccine were injected into mice by intramuscular method.The lymphocyte transformation function of immuned mice was detected;In the different periods,the dynamic variation of blood T lymphocytes were assayed;PPV and PCV2 antibodies were measured too.The experimental results showed that the MDBK cells with the transfection restructuring plasmid could not only amplify the ORF2-VP2 and IFN-γ gene,but also produce green fluorescence which could be seen by Fluorescence Microscope.Expression of the specific protein was detected too.All of these were considered as a confirmation of biological activities of the expressed protein.From the seventh-day on,the splenic lymphocytes had obvious response to ConA in the group of mice immuned by pCI-IFN-γ.ORF2.VP2,which was higher than that of control and the group of mice immuned by pCI-ORF2.VP2;the number of CD3 ＋CD4 ＋,CD3 ＋CD8 ＋T lymphocytes were higher or significantly higher than that of control group and the group of mice immuned by pCI-ORF2.VP2;On the fourteenth day,the antibodies of PPV and PCV were detected.These results manifested that the pCI-IFN-γ.ORF2.VP2 effectively induced humoral and cellular immunity response in organism.

关 键 词：猪细小病毒 VP2基因 猪圆环病毒 ORF2基因 IFN-Γ基因 

分 类 号：S852.659.2[农业科学—基础兽医学]