猪IL2基因重组对PPV VP2-PCV2 ORF2真核质粒免疫原性增强的研究

Effects of recombination of swine IL2 gene on the immunogenic of the PPV VP2-PCV2 ORF2 eukaryotic expression plasmid

将猪IL2成熟肽基因、PPV VP2基因和PCV2 ORF2抗原多肽基因定向克隆至真核表达载体pCI-neo,构建重组质粒pCI-ORF2-VP2和pCI-IL2-ORF2-VP2,用脂质体介导法将其转染PK15细胞,采用RT-PCR法和间接免疫荧光法检测重组质粒在体外的表达情况。并以小鼠为动物模型,分别将pCI-IL2-ORF2-VP2质粒、pCI-ORF2-VP2质粒、pCI空载体、猪细小灭活苗和圆环亚单位苗通过肌注进行免疫,检测免疫小鼠脾脏淋巴细胞转化功能,外周血T淋巴细胞亚群的动态变化及血清抗体效价。结果表明,重组质粒在体外能正常表达,pCI-IL2-ORF2-VP2免疫小鼠脾脏淋巴细胞转化功能,CD4+、CD8+T淋巴细胞的数量和血清PPV/PCV2抗体的OD值在二免后均高于或显著高于pCI-ORF2-VP2对照组。结果表明,猪IL2基因重组能显著增强VP2/ORF2核酸疫苗诱导的免疫应答。

To constructed the expression plasmid pCML2-ORF2-VP2,the swine IL2 gene,PPV VP2 gene and PCV2 ORF2 truncated gene（47-207aa） were cloned into the eukaryotic expression vector pCI-neo.After transfected PK15 cells by lipofectamine,the expressed product was detected by RT-PCR and immunofluore assay.To study the immune effects of DNA vaccine in vitro and in vivo,mice were used as the animal model.The recombinant plasmid pCI-IL2 -ORF2-VP2,pCI-ORF2-VP2,the control plasmid pCI-neo,the PPV vaccine and PCV2 vaccine were immunized by intramuscular injection,,two times in the same dose and two weeks interval.Anti-PPV/PCV2 antibodies were measured by ELISA,lymphocyte proliferation activity was detected using MTT method and the dynamic monitoring of the T-lymphocyte subsets,respectively.The results showed that the fusion protein can express nomally.And the reactive of the splenic T lymphocytes to the ConA stimulation、immune function of inducing the CD4 ＋,CD8 ＋ T lymphocyte,the PPV/PCV2 antibody levels of the pCI-IL2-ORF2-VP2 group were significantly higher than the pCT ORF2-VP2 group at different periods.The result indicated that recombinant of IL2 gene can enhance the immune response induced by VP2/ORF2 DNA vaccine in mice.