羊口疮病毒F1L基因真核表达质粒构建及对小鼠的免疫原性

Construction of Orf virus F1L gene eukaryotic expression plasmid and its immunological properties in mice

构建羊口疮病毒F1L基因的真核表达质粒，并探讨其对小鼠的免疫原性。本试验采用PCR扩增pMD18TFIL克隆质粒的F1L基因并将其克隆至pVAX1载体中，构建真核表达质粒并转染至MDBK细胞，采用RT—PCR和间接免疫荧光检测F1L基因在MDBK细胞中的转录和表达。将构建的pVAX1-F1L、pVAX1空载体、PBS对照通过后腿肌肉注射的方式免疫KM系小鼠，通过间接ELISA、MTT和FACS法对该DNA疫苗的免疫效果进行评价。结果表明，成功构建了真核表达质粒pVAX1F1L，并能在MDBK细胞中获得较高水平的表达。免疫小鼠后诱导的特异性抗体水平、脾淋巴细胞增殖反应、CD4^＋、CD8^＋细胞百分比和IL-2、IFN-γ、IL-4细胞因子水平均高于pVAX1纽和PBS对照组。因此，制备的重组核酸疫苗免疫小鼠后能够诱导机体产生体液免疫和细胞免疫。

To construct eukaryotic expression plasmid of Orf virus FIL gene and study its immuno- genicity in mice. The FIL gene of pMD18T-F1L cloning plasmid was amplified by PCR and cloned into eukaryotic expression vector pVAX1 to construct recombinant plasmids pVAX1-FIL which was transferred into MDBK cells by liposome. The transcription and expression of recombinant plasmids in MDBK cells were detected by RT-PCR and indirect immunofluorescence. The KM mice were injected with pVAX1-F1L, pVAX1 plasmids vetor and PBS controls in the muscle of back legs,the immunogenicity of the vaccine was evaluated by indirect ELISA, MTT and FACS. The results showed that recombinant eukaryotic expression plasmids pVAX-F1L was successfully constructed,and could be expressed with high leval in MDBK cells. Immunized mice presented spe- cific antibody level, lymphocyte proliferative response, CD4＋ , CD8＋ cell and IL-2, IFN-γ, IL-4 cyto- kines were significantly higher than those of pVAXl and PBS controls. Therefore, the recombinant vaccine can induce mice to produce humoral and cellular immunity.