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

发布时间：2018-04-27 15:24

本文选题：猪IFN-α + 口蹄疫病毒　；参考：《中国畜牧兽医》2017年05期

【摘要】：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-P12X3CIFN-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。
[Abstract]:In order to investigate the feasibility of co-expressing eukaryotic plasmids of foot-and-mouth disease virus (FMDV) polygene and porcine interferon 伪 (IFN- 伪) gene into clinical trials, the P12A3C and some 2B genes of foot-and-mouth disease virus (FMDV) P12X3C) and porcine IFN- 伪 gene were amplified by PCR. The recombinant plasmid pBudCE4.1-P12X3C-IFN- 伪 was transfected into BHK-21 cells to observe the expression of the target gene, and the recombinant plasmid was immunized to guinea pigs. Neutralizing antibody titer and T lymphocyte proliferation. The results showed that the recombinant plasmid pBudCE4.1-P12X3C-IFN- 伪 was successfully constructed by restriction endonuclease digestion and DNA sequence analysis. After transfection of pBudCE4.1-P12X3C-IFN- 伪, the recombinant plasmid was confirmed by Western blotting and indirect immunofluorescence assay. Compared with the recombinant plasmid pBudCE4.1-P12X3C, the recombinant plasmid pBudCE4.1-P12X3C-IFN- 伪 could induce a higher level of serum antibody against FMDV, and the titer of neutralizing antibody was higher than that of pBudCE4.1-P12X3C group. The lymphocyte proliferation of the recombinant plasmid pBudCE4.1-P12X3C-IFN- 伪 was 15%, while that of the recombinant plasmid pBudCE4.1-P12X3C group was 11%. The protective rate of recombinant plasmid pBudCE4.1-P12X3C-IFN- 伪 and inactivated vaccine group was 100, higher than that of recombinant plasmid pBudCE4.1-P12X3C group. In this experiment, the recombinant plasmid pBudCE4.1-P12X3CIFN- 伪 was successfully constructed. Pig IFN- 伪 as adjuvant can effectively assist DNA vaccine of foot-and-mouth disease to improve the immune response in vivo.
【作者单位】：中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室国家口蹄疫参考实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心;
【基金】：十三五国家重点研发计划(2016YFD0501503) 甘肃省科技支撑计划(1604NKCA045-2) 国家生猪现代产业技术体系项目(CARS-36-06B)
【分类号】：S852.65

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