联合表达PCV2 ORF2和PPV VP2基因病毒样颗粒获得及免疫原性  

Obtaining of union expression virus-like particle of PPV VP2 and PCV2 ORF2 and its immunogenicity

作　　者：徐志文[1] 郭万柱[1] 陈杨[1] 朱玲[1] 陈燕凌[1] 王印[1] 

机构地区：[1]四川农业大学动物生物技术中心,四川雅安625014

出　　处：《中国兽医学报》2009年第7期821-825,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30500019);四川省青年科技基金资助项目

摘　　要：将包含有完整闼读框架，长1740、705bp的PPVVP2基因、PCV2ORF2基因分别插入真核表达载体pPI-2．EGFP，构建了重组质粒pPi-Z．EGFP．VP2．ORF2。观察由脂质体介导转染重组质粒后的Vero细胞，结果最早在转染后20h左右出现荧光，36h达到高峰；对转染细胞进行电镜检测，结果观察到病毒样颗粒存在。为证实所获病毒样颗粒是否为重组颗粒，将纯化后的病毒样颗粒免疫仔猪，检测其血中T淋巴细胞的动态变化以及血清抗体水平。结果免疫仔猪的CD3＋、CD4＋T淋巴细胞在外周血淋巴细胞中的比率均有一定程度的升高；CD8＋T淋巴细胞在免疫后7～14d有所下降，之后再升高。抗体检测结果表明，免疫动物血清中有较高水平的PPV、PCV2特异性抗体。结果表明重组质粒pPI-2．EGFP．VP2．ORF2获得成功表达并形成重组病毒样颗粒，且该颗粒具有良好的免疫原性。The amplified VP2 gene （PPV strain SC-1） and PCV20RF2 gene were inserted into the eukaryotic expression vector pPI-2. EGFP. Then the recombinant plasmid named pPI-2. EGFP. VP2. ORF2 was obtained. Medi- ated by liposome, the recombinant plasmid was transfected into Veto cells and expressed. Using immunofluorescence assay,the fluorescence of expression products were first detected at 20 h after transfection and peaked 36 h. Under electronmicroscope,virus-like particles （VLPs） can be observed in the transfected cells. To confirm the obtained VLPs to be recombinant particles, piglets were immunized using purified VLPs. The dynamic variation of blood T lymphocytes and serum antibody level of PPV and PCV2 were measured. The results showed that the ratio of CD3＋ , CD4＋ T lymphocyte in peripheral blood lymphocyte of immunized piglets raised in a certain degree, the number of CDS＋T lymphocytes fell at 7-14 d after immunization,and then raised. Relatively high level of PP.V, PCV2 specific antibody could be detected. This indicated that the expression of recombinant plasmid pPI-2. EGFP. VP2. ORF2 was successful,the virus-like particles were formed and showed favourable immunogenicity.

关 键 词：病毒样颗粒 猪细小病毒 猪圆环病毒 免疫原性 

分 类 号：S852.659.2[农业科学—基础兽医学]