水生生物学报Issue(4)：751-757,7.DOI:10.7541/2015.98
草鱼呼肠孤病毒衣壳蛋白VP7真核重组质粒的构建及免疫效果评价
THE CONSTRUCTION OF PEGFP-N1-VP7 CONTAINING GCRVVP7 GENE AND STUDIES ON ITS IMMUNE EFFICACY
摘要
Abstract
In this study, to research and development the gene vaccine for Grass carp reovirus (Grass carp reovirus, GCRV), the recombinant plasmid pEGFP-N1-VP7 which carried the gene of the major capsid proteinVP7 of GCRV was constructed and identified. Then the recombinant plasmid was transfected into COS-1 and CIK cells with lipofectamine 2000, and the fluorescence microscope and RT-PCR were used to detect the transient expression. The results showed that the 830 bp fragment ofVP7gene was successfully transfected and expressed in COS-1 and CIK cells. To evaluate the immune efficacy of the DNA vaccine, the plasmids were extracted and purified from the mass culture of the recombinant bacteria containing the recombinant plasmid pEGFP-N1-VP7. Vaccine was diluted into 0.5 µg/100 µL and 5 µg/100 µL with PBS. Immune experiment was divided into four groups that are 0.5 µg group, 5 µg group, empty plasmid group and PBS control group. They were injected intramuscularly grass carp which weighs (20±5) g. Lymphocytes from different tissues and organs, such as the peripheral blood, the mesentery, the spleen and the kidney, were collected on Day 22 after the vaccination. The respiratory burst function was measured with NBT and the lymphocyte proliferation assay was conducted with MTT. The lymphocyte function of grass carps was up-regulated after the immunization with the DNA vaccine. In addition, the blood samples were also collected on Day 21, 28, 35, 42, 56, 70, 84 and 98 after the immunization. The specific antibody against VP7 in the sera of grass carps was detected with the triple antibody sandwich ELISA. These results demonstrated that the DNA vaccine could evoke both the cellular and the humoral immune response. The grass carps were immunized with 0.5 µg recombinant plasmids per tail, and the protection rate could reach 67%. Our study provided fundamental information on the research of the gene vaccine of GCRV.关键词
草鱼呼肠孤病毒/衣壳蛋白/真核表达/体液免疫/细胞免疫Key words
Grass carp reovirus/Major capsid protein/Eukaryotic expression/Humoral immunity/Cellular immunity分类
农业科技引用本文复制引用
郝贵杰,袁雪梅,潘晓艺,徐洋,姚嘉赟,蔺凌云,尹文林,沈锦玉..草鱼呼肠孤病毒衣壳蛋白VP7真核重组质粒的构建及免疫效果评价[J].水生生物学报,2015,(4):751-757,7.基金项目
浙江省科技厅重大科技专项重点农业项目(2012C12009-4) （2012C12009-4）
湖州市科技攻关计划项目(2011GG11) （2011GG11）
浙江省公益技术研究农业项目(2012C22046)资助 （2012C22046）
