猪圆环病毒核酸疫苗pcDNA-PCV-ORF2-ISS的构建及分子佐剂联合免疫研究  

The research on the construction of PCV2 DNA vaccine(pcDNA-PCV-ORF2-ISS) and combined immunization with molecular adjuvants

作　　者：张建远[1] 王红宁[1,2] 陈锦锐[2] 高荣[2] 黄勇[1] 罗昊[2] 陈建林[2] 郑敏[2] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]四川大学生命科学学院动物疾病防控生物工程研究中心,四川成都610064

出　　处：《中国兽医学报》2009年第12期1511-1515,共5页Chinese Journal of Veterinary Science

摘　　要：用PCR方法扩增猪圆环病毒PCVwhn株编码CAP蛋白的基因ORF2,同时人工合成一段CpG序列,并设计上下游引物,进行扩增,定向插入到真核表达载体pcDNA3.1(+)中,构建新型核酸疫苗(pcDNA-PCV-ORF2-ISS),并进行了酶切和测序鉴定。用构建的pcDNA-PCV-ORF2-ISS质粒以及pcDNA-PCV-ORF2与5种细胞因子分子佐剂质粒(IL-6/4、IL-2、IL-4、IL-6、IFN-γ真核表达质粒)联合对仔猪进行免疫试验。免疫后用间接ELISA法测定猪圆环病毒Ⅱ型特异性抗体lgG,用流式细胞仪测定猪血液CD3+、CD4+和CD8+T细胞亚类的数量。结果显示,成功构建含CpG免疫刺激序列的猪圆环病毒Ⅱ型核酸疫苗;pcDNA-PCV-ORF2-ISS在仔猪免疫实验能诱导细胞免疫并产生特异性抗体(P<0.05);用5种细胞因子分子佐剂质粒与pcDNA-PCV-ORF2疫苗同时免疫能显著提高DNA疫苗的免疫效果(P<0.05)。分子佐剂免疫增强效果次序为:IL-6、CpG、IL-6/4、IL-2、IL-4和IFNγ-,表明IL-6和CpG最佳。Amplified the ORF2 gene coding CAP protein in the strains of PCV-whn by PCR method. Synthesized a CpG oligodeoxynuleotide （ODN）, designed the primers and amplified it . Then inserted the results of the amplification in the pcDNA3.1（＋）, and constructed a novel DNA vaccine（pcDNA-PCV-ORF2-ISS）checked by sequencing . Piglets were immunized with the vaccine and five kind of cytokines （IL-6/4, IL-2, IL-4, IL-6, IFN-γ）. After that, the specific antibody were detected by the ELISA assay,and the number of CD3＋, CD4＋,CD8 ＋ T lymphocytes were counted by Fluorescence Activated Cell Sorter（FACS）. The results were as follows： The specific antibodies to the PCV2 mounted significantly in the sera from the pcDN-PCV-ORF2-ISS vaccinated piglets（P〈0.05）. And the pcDNA-PCV-ORF2-ISS DNA vaccine could remarkably provoked the proliferation of CD3 ＋, CD4＋, CD8 ＋ T-lymphocytes of pig in vitro in comparison with the control （P〈0.05）. The immune efficiency of the molecular adjuvants in order of immune enhancement were IL-6, CpG, IL-6/4, IL-2, IL- 4 and IFN-γ, respectively, which demonstrated that IL-6 and CpG were the best.

关 键 词：PCVⅡ型 DNA疫苗 CpG免疫刺激序列 细胞因子分子佐剂 质粒 

分 类 号：S852.65[农业科学—基础兽医学]