鸡痘病毒抑制宿主细胞凋亡SERPIN基因的筛选及活性分析

Serine protease inhibitors (SPI) is exists widely in mammals, plants and microorganisms. It has been reported that a variety of poxviruses encodes SPI gene, including SPI-1, SPI-2 / CrmA and SPI-3. SPI-1 has a role in the decision host range, SPI-2 could inhibits apoptosis and inflammatory response of host and so on. By sequencing the whole genome of the fowlpox virus (FPV) 282E4, and then we found five SERPIN genes. The project was based on the research results of poxviridae to establish a DF1 cell lines that could stable expression the SERPIN protein. Then we screening the SERPIN that inhibit host apoptotic activity by morphological observation, cell activity detected with MTT, Caspase-3、8、 activity assay and flow cytometry. We further build the recombinant FPV of missing SERPIN, to verification apoptotic activity by inoculated DF1, DF1/SERPIN cell with FPV△SERPIN. Further we utilizing Co-immunoprecipitation experiments identified protein that interactions with SERPIN. In summary, this result can provide experimental data for the biology function of the SERPIN protein of avian pox virus.

丝氨酸蛋白酶抑制剂（serine protease inhibitors，SPI）广泛存在于哺乳动物、植物和微生物体内。多种痘病毒编码SPI，其中正痘病毒属编码的SPI包括SPI-1、SPI-2/CrmA、SPI-3。SPI-1具有决定宿主范围的作用，SPI-2具有抑制宿主炎症反应和细胞凋亡等功能。进行鸡痘病毒（fowlpox virus，FPV）282E4全基因组测序及序列分析发现其基因组中存在5个SERPIN基因，本研究拟根据痘病毒科现有研究基础，建立稳定表达SERPIN蛋白的DF1细胞系，通过形态学观察、MTT检测细胞活性、Caspase活性检测、流式细胞术等筛选出具有抑制宿主细胞凋亡活性的SERPIN，进一步构建缺失SERPIN的重组FPV接种DF1、DF1/SERPIN细胞进行验证，免疫共沉淀鉴定与SERPIN相互作用的蛋白，为禽痘病毒SERPIN蛋白生物学功能研究提供实验数据。

本研究克隆鸡痘病毒SERPIN基因并进行原核表达，以FPV基因组为模板，PCR扩增SERPIN基因克隆至表达载体pET32a，构建重组表达质粒pET32a-SORF3，将重组质粒转化至感受态细胞BL21中，IPTG诱导表达并进行SDS-PAGE及Werstern-blot鉴定。结果构建的原核表达质粒pET32a-SERPIN转化BL21后经IPTG诱导4 h，可表达46-56KD融合蛋白，Western blot显示重组蛋白可以被Anti-His标签抗体识别，表明成功构建了pET32a-SERPIN原核表达质粒，并能在大肠杆菌中表达具有免疫反应性的SORF3。设计荧光定量PCR检测引物，建立检测标准曲线，构建真核表达质粒pCAGEN-SERPIN。设计合成穿梭载体pTS2GFPV264，转染CEF经15-20次噬斑筛选，PCR鉴定阳性重组病毒rFPV-264。为进一步了解禽痘病毒的生物学特性、致病机理研究提供理论基础，并对深入认识和拓展理解SERPIN的作用具有重要的科学意义。