山羊痘病毒类N1L基因毒力作用机制的研究

Goat pox (GTP) is highly contagious and infectious animal disease. Goatpox virus (GTPV) AV41 strain attenuated vaccine is developed by China and widely used to prevent animal from GTPV. However, it still has residual virulence, causing serious adverse reactions such as abortion in some breed line of animals. GTPV and vaccinia virus are poxviridae. Previous researches have shown that there are abundant virulence and immunosuppressive factors in pox viruses, among which N1L is the strong virulence factor and could inhibit apoptosis and NF-κB activation, which is required for viral virulence. However, it is rarely reported in GTPV. The applicant had conducted a preliminarily investigation about NIL-like gene (N1L) in AV41 and found that N1L could effectively inhibit NF-κB signaling pathway and apoptosis. Therefore, the applicant will try to clarify the detail mechanism of N1L evading host innate immune response and viral virulence. After that, the AV41 containing deletion of N1L gene or mutants N1L impaired for NF-κB or apoptosis inhibition, plasmids expressing GTPV N1L or GTPV N1L impaired for NF-κB or apoptosis inhibition and VV N1L substituted for GTPV N1L recombinant will be constructed. All the data will demonstrated the function and virulence mechanisms of GTPV N1L, which will fill a gap of study on GTPV virulence, and offer a theoretical basis for goat poxvirus vaccine in future.

山羊痘为高度接触性烈性传染病。我国自主研发的山羊痘AV41减毒疫苗株，被广泛应用于山羊痘的免疫预防。但该疫苗株仍有一定的毒力残留，表现为某些品种或品系的绵羊、山羊接种后副反应严重。山羊痘病毒（GTPV）与痘苗病毒（VV）均属痘病毒科，在痘病毒庞大的基因组中存在很多与毒力相关的基因，现有研究表明，VV中的N1L基因是较强的毒力因子，能够抑制细胞凋亡和抑制NF-κB信号通路，且毒力作用与其抑制NF-κB信号通路密切相关。但山羊痘病毒还没有相关的报道。申请者对AV41疫苗株基因组中类N1L基因（简称N1L基因）的初步研究表明，它与VV中的N1L具有相似的结构和功能，都能够有效地抑制NF-κB的激活和细胞凋亡。为此，本申请人将以GTPV N1L基因为研究对象，拟阐明该基因的功能及其毒力作用机制，填补羊痘病毒中毒力相关基因的研究空白，为研制安全有效的山羊痘疫苗提供新的理论依据。

山羊痘为高度接触性烈性传染病。我国自主研发的山羊痘AV41减毒疫苗株，被广泛应用于山羊痘的免疫预防。但该疫苗株仍有一定的毒力残留，表现为某些品种或品系的绵羊、山羊接种后副反应严重。山羊痘病毒（GTPV）与痘苗病毒（VV）均属痘病毒科，在痘病毒庞大的基因组中存在很多与毒力相关的基因，现有研究表明，VV中的N1L基因是较强的毒力因子，能够抑制细胞凋亡和抑制NF-κB信号通路，且毒力作用与其抑制NF-κB信号通路密切相关。但山羊痘病毒还没有相关的报道。. 本研究主要是以山羊痘病毒N1L（135）基因为研究对象，构建了表达GTPV N1L蛋白的真核表达质粒以及利用CRISPR/Cas9基因敲除技术结合传统的插入报告基因EGFP，构建N1L缺失的重组山羊痘病毒。在细胞水平上研究N1L基因能否抑制NF-κB信号通路以及能否抑制细胞凋亡及其作用机制；用N1L缺失的重组山羊痘病毒接种山羊来研究N1L蛋白对山羊痘疫苗株AV41毒力的影响；通过构建缺乏抑制NF-κB作用和缺乏抑制凋亡作用的GTPV N1L蛋白的突变体（真核表达质粒）和相应的GTPV N1L突变毒株，通过感染或者转染细胞以及接种山羊来揭示其毒力作用机制，进而增加对山羊痘病毒免疫调节蛋白与宿主相互作用的理解，为以后的山羊痘疫苗研制提供新的指导和理论依据。本文证实了GTPV 135基因能够抑制NF-κB信号通路的激活能够抑制细胞凋亡。135基因较TK基因对病毒复制的影响更小，而TK 和135两种插入位点作比较，达外源蛋白(PPRV H)，135插入位点能够诱导更强的体液免疫反应。