重组口蹄疫鸡痘病毒vUTAL3CP1的构建及其遗传稳定性和免疫原性

Construction,Genetic Stability and Immunogenicity of Recombinant Fowlpox Virus vUTAL3CP1 with Capsid Protein Precursor P1-2A of Foot and Mouth Disease Virus

目的构建重组口蹄疫鸡痘病毒,并检测其遗传稳定性和免疫原性。方法将口蹄疫病毒(FMDV)的衣壳蛋白前体P1-2A和蛋白酶3C基因插入到鸡痘病毒表达载体中,构建重组鸡痘病毒转移载体pUTAL3CP1,并与鸡痘病毒(FPV)282E4株共转染鸡胚成纤维细胞(CEF),通过BrdU加压筛选获得重组鸡痘病毒vUTAL3CP1。重组病毒在CEF中连续传30代,分别进行毒力和基因检测,并免疫BALB/c小鼠,检测特异性抗体和中和抗体。结果重组鸡痘病毒经RT-PCR可扩增出目的基因;特异性荧光抗体反应呈阳性;在CEF中连续传30代,毒力稳定,基因未发生缺失和变异;免疫小鼠能产生较高水平的FMDV特异性抗体和中和抗体。结论已成功构建重组口蹄疫鸡痘病毒,且具有良好的遗传稳定性和免疫原性。

Objective To construct recombinant fowlpox virus （FPV） with foot and mouth disease virus （FMDV） and study its genetic stability and immunogenicity. Melhods Insert the genes encoding capsid protein precursor P1-2A and protease 3C into FPV expression vector. Co-transfect chick embryo fibroblast （CEF） with the constructed recombinant plasmid pTUAL3CP1 and FPV 282E4 strain to obtain recombinant fowlpox virus vUTAL3CP1 by BrdU pressure screening. The vUTAL3CP1 was subcttltured in CEF for 30 passages and tested for virulence and genetic stability. Immunize BALB/c mice with vUTAL3CP1 and determine serum specific antibody and neutralizing antibody. Results The tar- get gene was amplified from vUTAL3CP1 by RT-PCR. IFA showed positive reaction of vUTAL3CPl-transfected CEF with specific antibody against FMDV.The virulence of vUTAL3CP1 was stable after subculture in CEF for 30 passages, and no deletion or variation of gene was observed. High titers of specific antibody and neutralizing antibody were induced in mice immumized with vUTAL3CP1. Condusion Recombinant fowlpox virus vUTAL3CP1 was successfitlly constructed and showed good genetic stability and immunogenicity.