口蹄疫病毒多基因及猪α干扰素共表达载体的构建及其免疫豚鼠效果研究

为探讨口蹄疫病毒多基因及猪α干扰素（IFN-α）基因共表达真核质粒进入临床试验的可行性，本试验用PCR方法扩增了口蹄疫病毒P12A3C及部分2B基因（P12X3C）和猪IFN-α基因，克隆到真核表达载体pBudCE4.1中，经双酶切鉴定后，将重组质粒pBudCE4.1-P12X3C-IFN-α转染BHK-21细胞中，观察目的基因的表达，并将重组质粒免疫豚鼠，检测豚鼠的血清抗体水平、中和抗体滴度及T淋巴细胞增殖情况。结果显示，经酶切鉴定及DNA序列分析成功构建了重组质粒pBudCE4.1-P12X3C-IFN-α，转染BHK-21细胞后，通过Western blotting、间接免疫荧光试验鉴定证实重组质粒能有效表达。ELISA结果显示，重组质粒pBudCE4.1-P12X3C-IFN-α比重组质粒pBudCE4.1-P12X3C能诱导机体产生更高水平的抗口蹄疫病毒的血清抗体，且中和抗体滴度也高于重组质粒pBudCE4.1-P12X3C组。MTT法检测结果表明，重组质粒pBudCE4.1-P12X3C-IFN-α组淋巴细胞增殖可达15%，而重组质粒pBudCE4.1-P12X3C组则为11%。攻毒后重组质粒pBudCE4.1-P12X3C-IFN-α组和灭活疫苗组保护率达100%，高于重组质粒pBudCE4.1-P12X3C组的80%。本试验成功构建了重组质粒pBudCE4.1-P12X3C-IFN-α，猪IFN-α作为佐剂可有效辅助口蹄疫DNA疫苗提高动物体内的免疫反应。

Construction of Co-expression Vector Containing Multi-gene of FMDV and Porcine IFN-α and Its Immunogenicity Effect in Guinea Pig

In order to explore the feasibility of entering clinical trials of multi-gene of foot-and-mouth disease virus (FMDV) and porcine interferon-α(IFN-α) co-expression plasmids, porcine IFN-α and the multi-gene of FMDV that included full length of P12A3C,and part of 2B (P12X3C) were amplified by PCR and cloned into the eukaryotic expressing plasmid pBudCE4.1 to construct the recombinant vector. After identification by restriction enzyme digestion, this vector was transfected into the BHK-21 cell and its expression were observed by Western blotting and indirect fluoroscopy assay.The serum antibodies, neutralizing antibody titers and T-lymphocyte proliferation were detected in guinea pigs after immunization of the recombinant vector. The results showed that the recombinant plasmid pBudCE4.1-P12X3C-IFN-α was successfully constructed after detecting by enzyme digestion and sequencing. Recombinant plasmid was effectively expressed in BHK-21 cells after detecting by Western blotting and indirect fluoroscopy assay. Result of ELISA indicated that anti-FMDV IgG antibody level of pBudCE4.1-P12X3C-IFN-α group was higher compared with pBudCE4.1-P12X3C group.Similar results for serum neutralization titers were obtained. MTT assay showed that proliferation of lymphocyte was 15% in pBudCE4.1-P12X3C-IFN-α group and 11% in case of pBudCE4.1-P12X3C group. After challenged with homologous virus, protection rate of conventional vaccine and recombinant plasmid pBudCE4.1-P12X3C-IFN-α group were 100%, while the recombinant plasmid pBudCE4.1-P12X3C group was 80%.The present study showed the recombinant plasmid pBudCE4.1-P12X3C-IFN-α was successfully constructed, and could markedly improve the efficacy of DNA vaccine against FMDV.