融合表达猪圆环病毒2型ORF1和ORF2基因真核质粒的构建及对小鼠的免疫原性

Construction of porcine circovirus type 2 ORF1 and ORF2 fusion genes eukaryotic expression vector and its immunological properties in mice

用PCR法扩增出猪圆环病毒2型的Rep蛋白基因(933bp)和Cap蛋白基因(705bp),将其定向克隆于真核表达载体pcDNA3.1(+)的多克隆位点上,构建真核表达质粒pcDNA-ORF1-ORF2。将构建好的重组质粒pcD-NA-ORF1-ORF2按100μg/只腿部肌肉注射BALB/c小白鼠,同时设pcDNA-ORF1、pcDNA-ORF2、pcDNA3.1-(+)、PCV2全毒疫苗和PBS为对照,共免疫2次,间隔2周。分别于首免后第0、7、14、21、28、42天用MTT法检测小鼠脾淋巴细胞的增殖效应,用ELISA法检测小鼠的抗体水平;并于首免后第0、7、14、21、28天测定脾淋巴细胞中各细胞亚群的比例,对该核酸疫苗的免疫原性进行初步评价。结果显示,重组质粒能诱导鼠体产生较强的细胞免疫和体液免疫,并从免疫后第7天起所测各组数据均显著高于(P<0.05)或极显著高于(P<0.01)其他试验组。结果表明,将ORF1和ORF2基因共同用于PCV2核酸疫苗的研发具有很好的前景,为研究新型猪圆环病毒疫苗奠定了基础。

The Rep protein and Cap protein gene of porcine circovirus type2 were amplified by PCR technique and cloned into multiple cloning sites with eukaryotic expressing vectors pcDNA3.1（＋） to construct nucleotide vaccine plasmid pcDNA-ORF1-ORF2.The constructed pcDNA-ORF1-ORF2 was developed into nucleotide vaccine and intramuscularly inject it into BALB/c mice with 100 μg at two weeks interval.In the same time,the pcDNA-ORF1 vaccine,pcDNA-ORF2 vaccine,PCV2 vaccine,pcDNA3.1（＋） empty vector and PBS were as the control groups.to evaluate the pcDNA-ORF1-ORF2 vaccine immunogenicity.On the first,7th,14th,21th,28th and 42th day after the first vaccination we detected sera antibody using ELISA and calculated mice spleen T lymphocytes subgroups,and also the cell mediated immunity by MTT.The results indicated that the pcDNA-ORF1-ORF2 vaccine was able to induce satisfactory humoral immunity and cellular immunity which were significantly higher than other control groups after day 7.In conclusion,The ORF1 gene with ORF2 gene used for developing the DNA vaccines against porcine circovirus type 2 laid the foundation of the research on this field.