PPV VP2基因和PCV2 ORF2抗原优势区基因真核表达质粒的构建及其免疫效果

Construction of the eukaryotic expression plasmid with PPV VP2 gene and PCV2 ORF2 antigen predominance region gene and study on immunogenicity of the plasmid

采用PCR法扩增猪圆环病毒2型(PCV2)ORF2抗原优势区(ORF2′)基因和猪细小病毒(PPV)VP2基因,将目的基因定向插入真核表达载体pCI-neo,构建了重组质粒pCI-ORF2′-VP2。将重组质粒免疫小鼠,同时设立PCV亚单位疫苗、PPV灭活疫苗和空载体对照组,采用MTT比色法、流式细胞术和ELISA法分别对免疫小鼠脾淋巴细胞的转化功能,外周血CD4+和CD8+T淋巴细胞比例,PCV2和PPVIgG抗体效价进行了检测。结果表明,从免疫后第7d起,pCI-ORF2′-VP2免疫小鼠脾淋巴细胞的增殖活性,外周血CD4+、CD8+T淋巴细胞比例和抗PCV2、PPV的特异性抗体效价都显著(P<0.05)或极显著(P<0.01)高于空载体对照组,且重组质粒组在第21～42d诱导的免疫水平显著或极显著强于PPV灭活疫苗组。证实,构建的重组质粒pCI-ORF2′-VP2能够诱导小鼠产生良好的细胞免疫和体液免疫应答。

Porcine parvovirus（PPV） VP2 gene and porcine circovirus type 2 （PCV2） ORF2 antigen predominance region（ORF2＇） gene （from 157 to 183aa in Cap protein） were amplified,and then cloned into the pCI-neo vector to construct the eukaryotic expression plasmid pCI-ORF2＇-VP2.Mice were inoculated with pCI-ORF2＇-VP2 and PPV vaccine,PCV vaccine and empty vector were developed as controls.The immune efficacy was determined by the MTT assay,FCM and ELISA,respectively.In result,the reaction of the splenic T lymphocytes to ConA stimulation,the percentages of CD4＋ and CD8＋ T lymphocyte,the levels of the antibodies against PPV and PCV2 in the pCI-ORF2＇-VP2 group were significantly higher than those in the empty vector group from day 7 on post-inoculation.The immune levels resulted from the recombinant plasmid was higher than that from the PPV vaccine.The results indicated that pCI-ORF2＇-VP2 was able to induce satisfactory humoral and cellular immune responses.