猪静脉血管内皮细胞cDNA文库构建及与猪非典型瘟病毒云南株关键变异基因互作蛋白的筛选  

Construction of SVEC cDNA Library and Screening of Interacting Proteins with Key Variant Gene of APPV-YN

作　　者：姜光飞 何维琪 余蕊 梁龙舟 孙永科 杨玉艾 JIANG Guang-fei;HE Wei-qi;YU Rui;LIANG Long-zhou;SUN Yong-ke;YANG Yu-ai(College of Veterinary Medicine,Yunnan Agricultural University,Kunming,Yunnan,650201,China)

机构地区：[1]云南农业大学动物医学院,云南昆明650201

出　　处：《动物医学进展》2023年第7期1-9,共9页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(32160485)。

摘　　要：近几年,猪非典型瘟病毒(APPV)在世界多个养猪国家相继报道,临床症状呈不典型性,给养猪业造成了较大的经济损失。为了解猪非典型瘟病毒云南株关键变异基因(APPV-YN Mut)与宿主可能存在相互作用的蛋白,深入研究APPV-YN Mut基因的功能及其非典型化机制,利用酵母双杂交技术,构建了猪静脉血管内皮细胞(SVEC)cDNA文库,并进一步采用建立的SVEC cDNA文库与诱饵质粒pGBKT7-Mut共转Y2H Gold酵母菌,筛选与Mut蛋白相互作用的蛋白,对筛选出的蛋白进行测序和生物信息学分析。结果表明,构建的诱饵载体在酵母细胞中成功表达,且无毒无自激活;构建的SVEC cDNA文库容量大于1×106 CFU/mL,文库滴度6.2×106 CFU/mL,PCR显示文库插入片段大小在250~3000 bp,条带大小不一,符合文库大小要求;与SVEC cDNA文库筛选发现14种与Mut蛋白存在潜在互作的宿主蛋白,通过蛋白功能分析发现这些蛋白主要与病毒复制、细胞凋亡、钙离子结合、蛋白代谢和降解有关。研究结果有助于进一步探索14种潜在互作蛋白中到底哪些与Mut发生密切互作,且与病毒感染宿主密切相关,为研究APPV-YN Mut基因功能及其致非典型化作用机制奠定了理论基础。In recent years,atypical swine fever virus(APPV)has been reported in many pig-raising countries in the world,and the clinical symptoms are atypical,causing certain economic losses to the pig industry.In order to understand the proteins that may interact with the host of the key variant gene of the Yunnan strain of atypical pestilence virus(APPV-YN Mut),the function of the APPV-YN Mut gene and its atypia mechanism were further studied.In this study,a porcine venous vascular endothelial cell(SVEC)cDNA library was constructed by using yeast two-hybrid technology,and the established SVEC cDNA library was further used to co-transfect Y2H Gold yeast with the bait plasmid pGBKT7-Mut to screen proteins that interact with Mut protein.Sequencing and bioinformatics analysis of the screened proteins were conducted.The results showed that the constructed bait vector was successfully expressed in yeast cells,and was non-toxic and non-self-activating;the capacity of the constructed SVEC cDNA library was greater than 1×106 CFU/mL,and the library titer was 6.2×106 CFU/mL.PCR showed that the size of the library insert was 250-3000 bp,the band size varies,which meets the library size requirements;14 host proteins potentially interacting with the Mut protein were found by screening with the SVEC cDNA library.It is involved in ion binding,protein metabolism and degradation.The results of this study help us to further explore which of the 14 potential interacting proteins closely interact with Mut and are closely related to the virus-infected host,laying a solid foundation for the study of APPV-YN Mut gene function and its atypia-causing mechanism.theoretical basis.

关 键 词：猪非典型瘟病毒 关键变异基因Mut 酵母双杂交 CDNA文库 蛋白互作 

分 类 号：S852.629.6[农业科学—基础兽医学] S858.28[农业科学—兽医学]