【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。更多还原

【Abstract】 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(IL6/4,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 pcDNAPCV-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.更多还原