含T细胞表位的猪细小病毒VP2基因核酸疫苗的构建及其在小鼠中的免疫原性分析

Construction and immunogenicity of nucleic acid vaccine expressing porcine parvovirus VP2 fused with porcine circovirus T cell epitope

为研制猪细小病毒(PPV)主要保护性抗原VP2基因核酸疫苗,本研究将猪圆环病毒(PCV)编码T细胞表位P21多肽的序列连接于PPV VP2基因的5'端克隆于pcDNA3.1(+)中,构建重组表达质粒pcDNA-P21-VP2,并将其经磷酸钙介导转染HEK293T细胞中检测其表达情况。SDS-PAGE和western blot检测表明,P21-VP2重组蛋白获得了有效的表达,其分子量约为67 ku。将pcDNA-P21-VP2肌肉注射BALB/c小鼠后,采用ELISA方法检测其抗体,并采用MTT法检测小鼠免疫后脾脏淋巴细胞的增殖活性。试验结果表明,重组质粒能够有效诱导小鼠产生明显的抗体反应,并且对淋巴细胞的增殖反应有明显促进作用。该实验结果为研制有效的PPV核酸疫苗奠定了基础。

To investigate the DNA vaccine against porcine parvovirus （PPV）, the helper T-cell epitope （P21） encoding sequence was fused on 5＇end of PPV VP2 gene and cloned into pcDNA3.1 （＋） to construct the recombinant plasmid of pcDNA-P21-VP2. The expression of the recombinant protein P21-VP2 was verified in pcDNA-P21-VP2 transfected HEK293T cells by western blot. In addition, mice were immunized by intramuscular injection of pcDNA-P21-VP2 at dosage of 10 μg three times at 2-week intervals and the results showed that the antibody titer was the same in mice induced by pcDNA-P21-VP2 as the PPV inactivated vaccine detected by ELISA. Furthermore, the MTT assay indicated that the spleen lymphocyte proliferation was significantly higher in recombinant plasmid group than that of control group. In conclusion, our data demonstrated the pcDNA-P21-VP2 was able to efficiently induce the humoral and cellular immune responses which provide a basis for developing a new efficient candidate DNA vaccine against PPV and PCV2.