IBV与IBDV二联核酸疫苗构建及与鸡IL-18共同免疫效力研究
发布时间：2018-10-09 07:25
【摘要】：本研究首先对IBV S1、IBDV VP2基因进行亚克隆,并利用一段linker将IBV S1、IBDV VP2基因连接后,利用真核表达载体pVAX-1,通过基因重组分别构建真核重组表达质粒pVAX-IBV S1、pVAX-IBDV VP2和pVAX-IBV S1-linker-IBDV VP2。体外瞬时转染实验表明,外源基因能够在细胞内进行表达,并且表达后的蛋白可以被相应的IBV S1多克隆抗体和IBDV VP2多克隆抗体识别,说明了插入的外源基因具备各自的免疫反应性,为动物体内试验提供了基础。 将试验鸡分为8组,即S1组(免疫pVAX-IBV S1真核重组质粒)、VP2组(免疫pVAX-IBDV VP2真核重组质粒)、SV组(免疫pVAX-IBV S1-linker-IBDV VP2真核重组质粒)、SV18组(免疫pVAX-IBV S1真核重组质粒和pVAX-ChIL-18真核重组质粒)、18组(免疫pVAX-ChIL-18真核重组质粒)、YM组(免疫IBVH120疫苗和IBDV疫苗)、PBS组(免疫PBS)、pVAX组(免疫pVAX空载体质粒)。免疫过程中利用淋巴细胞增殖试验、酶联免疫吸附试验、中和抗体试验以及利用流式细胞术检测CD4+、CD8+T淋巴细胞动态变化,对不同日龄的试验动物进行检测,最后用病毒攻击试验比较免疫不同质粒组别之间保护率。 实验结果表明,二联核酸疫苗对两种病毒的攻击有一定的保护作用,添加了ChIL-18作为免疫佐剂增强了二联核酸疫苗的免疫效果,同时增加了核酸疫苗对病毒攻击的保护力。
[Abstract]:In this study, we first subcloned the IBV S1G IBDV VP2 gene, then ligated the IBV S1 BDV VP2 gene with a piece of linker, then constructed the eukaryotic expression plasmids pVAX-IBV S1 pVAX-IBDV VP2 and pVAX-IBV S1-linker-IBDV VP2. by gene recombination using the eukaryotic expression vector pVAX-1,. Transient transfection in vitro showed that the foreign gene could be expressed in cells, and the expressed protein could be recognized by the corresponding IBV S1 polyclonal antibody and IBDV VP2 polyclonal antibody. The results showed that the inserted foreign genes had their own immunoreactivity, which provided the basis for in vivo experiments in animals. The chickens were divided into 8 groups. That is, S1 group (immunized with pVAX-IBV S1 eukaryotic recombinant plasmid) and VP2 group (immunized with pVAX-IBDV VP2 eukaryotic recombinant plasmid) and SV18 group (immunized with pVAX-IBV S1 eukaryotic recombinant plasmid and pVAX-ChIL-18 eukaryotic recombinant plasmid) and SV18 group (immunizing pVAX-ChIL-18 eukaryotic recombinant plasmid) and SV18 group (immunizing pVAX-IBV S1-linker-IBDV VP2 eukaryotic recombinant plasmid) and SV18 group (immunizing pVAX-IBV S1 eukaryotic recombinant plasmid and pVAX-ChIL-18 eukaryotic recombinant plasmid). The recombinant plasmids were divided into two groups: YM group (immunized with IBVH120 vaccine and IBDV vaccine) and PBS group (immunized with PBS) and pVAX group (immunized with pVAX unloaded particles). Lymphocyte proliferation test, enzyme-linked immunosorbent assay (Elisa), neutralizing antibody test and flow cytometry were used to detect the dynamic changes of CD8 T lymphocytes in different ages. Finally, virus attack test was used to compare the protective rate among different plasmid groups. The results showed that the binucleic acid vaccine had a certain protective effect against the two viruses, and the addition of ChIL-18 as an immune adjuvant enhanced the immune effect of the vaccine and increased the protection of the DNA vaccine against the virus attack.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2258472
[Abstract]:In this study, we first subcloned the IBV S1G IBDV VP2 gene, then ligated the IBV S1 BDV VP2 gene with a piece of linker, then constructed the eukaryotic expression plasmids pVAX-IBV S1 pVAX-IBDV VP2 and pVAX-IBV S1-linker-IBDV VP2. by gene recombination using the eukaryotic expression vector pVAX-1,. Transient transfection in vitro showed that the foreign gene could be expressed in cells, and the expressed protein could be recognized by the corresponding IBV S1 polyclonal antibody and IBDV VP2 polyclonal antibody. The results showed that the inserted foreign genes had their own immunoreactivity, which provided the basis for in vivo experiments in animals. The chickens were divided into 8 groups. That is, S1 group (immunized with pVAX-IBV S1 eukaryotic recombinant plasmid) and VP2 group (immunized with pVAX-IBDV VP2 eukaryotic recombinant plasmid) and SV18 group (immunized with pVAX-IBV S1 eukaryotic recombinant plasmid and pVAX-ChIL-18 eukaryotic recombinant plasmid) and SV18 group (immunizing pVAX-ChIL-18 eukaryotic recombinant plasmid) and SV18 group (immunizing pVAX-IBV S1-linker-IBDV VP2 eukaryotic recombinant plasmid) and SV18 group (immunizing pVAX-IBV S1 eukaryotic recombinant plasmid and pVAX-ChIL-18 eukaryotic recombinant plasmid). The recombinant plasmids were divided into two groups: YM group (immunized with IBVH120 vaccine and IBDV vaccine) and PBS group (immunized with PBS) and pVAX group (immunized with pVAX unloaded particles). Lymphocyte proliferation test, enzyme-linked immunosorbent assay (Elisa), neutralizing antibody test and flow cytometry were used to detect the dynamic changes of CD8 T lymphocytes in different ages. Finally, virus attack test was used to compare the protective rate among different plasmid groups. The results showed that the binucleic acid vaccine had a certain protective effect against the two viruses, and the addition of ChIL-18 as an immune adjuvant enhanced the immune effect of the vaccine and increased the protection of the DNA vaccine against the virus attack.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 于京穹;鸡传染性支气管炎病毒S1基因和M基因二价核酸疫苗构建与免疫效力研究[D];东北农业大学;2012年
，本文编号：2258472
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