真核共表达载体pCI-IFN-α-ORF2-VP2中IFN-α对其免疫原性的影响

Study on the immunogenicity effects of co-expression of IFN-α in recombinant plasmid pCI-IFN-α-ORF2-VP2

采用PCR技术克隆分析了太湖猪IFN-α基因(PoIFN-α)CDS区全序列,将去信号肽的PoIFN-α基因片段插入重组质粒pCI-ORF2-VP2(已构建)构建了真核共表达质粒pCI-IFN-α-ORF2-VP2,并肌肉注射免疫小鼠,检测了不同时期小鼠的PPV和PCV2抗体水平、脾淋巴细胞增殖功能和外周血T淋巴细胞亚群的变化情况。结果,扩增出891bp大小包括PoIFN-α全基因的特异条带;小鼠免疫试验显示,pCI-IFN-α-ORF2-VP2质粒免疫组第7天后脾淋巴细胞对ConA开始有明显反应,同时可检测到抗PPV和PCV2的抗体,第14天后脾淋巴细胞增殖反应和抗体水平均高于pCI-ORF2-VP2免疫组,CD4+T细胞值和CD8+T细胞值第7天开始高于pCI-ORF2-VP2和pCI-neo空载体试验组。提示,真核共表达载体pCI-IFN-α-ORF2-VP2中PoIFN-α基因对其诱导机体产生的细胞免疫和体液免疫有一定的增强作用。

The whole porcine IFN-α gene was amplified by PCR from the liver cell of the Taihu pig, and the signal-peptide-free PoIFN-α gene were cloned and inserted into the eukaryotic expression plasmid pCI- ORF2-VP2 （constructed）to construct the co-expression eukaryotic plasmid pCI-IFN-α-ORF2-VP2. Then the eukaryotic plasmid pCI-IFN-α-ORF2-VP2 were injected into mice by intramuscular method. The lymphocyte transformation function,antibodies against PPV and PCV2 and the dynamic variation of blood T lymphocytes in immunized mice in the different periods were examined by using MTT method, ELISA and flow cell meter（FCM）,respectively. The results showed that a 891 bp DNA fragment including whole CDS of PolFN-α gene was obtained and the eukaryotic plasmid pCI-IFN-α-ORF2-VP2 was constructed. Animal experiments showed that,on the 7th day,the splenic lymphocytes had response to ConA and specific antibodies against PPV and PCV2,could be detected in the group of mice immunized with pCI-IFN-α-ORF2-VP2, and their levels were higher than that of the group of mice immunized with pCI-ORF2-VP2 from the 14th day. The numbers of CD4^＋ T lymphocytes and CD8^＋ T lymphocytes were higher than that of control group immunized with pCI-neo vecter and the group immunized with pCI-ORF2-VP2. These results indicated coexpression of IFN-α in recombinant plasmid pCI-IFN-α-ORF2-VP2 may enhance vaccine effect of the recombinant plasmid on inducing humoral and cellular immunity response.