【作者】 杨倩；

【导师】 鲜思美；

【作者基本信息】 贵州大学 ， 兽医学硕士， 2022， 硕士

【摘要】 羊口疮（Orf）是由羊口疮病毒（OrfVirus,OrfV）引起的一种接触性、嗜上皮性人兽共患传染病。OrfV主要感染绵羊和山羊,羔羊的发病率可高达90%～100%,主要特征病变为口唇、鼻部等处形成增生性损伤,是危害养羊业的重要传染病。OrfV为痘病毒科（Poxviridae）、副痘病毒属（Parapoxvirus）成员,为有囊膜线性双股DNA病毒。F1L蛋白为病毒表面囊膜蛋白,研究发现F1L蛋白具有肝素结合活性,与病毒吸附和侵入宿主细胞有关,在病毒感染早期阶段起作用。羔羊睾丸（Lamb testicular,LT）细胞是OrfV增殖的敏感细胞,细胞膜表面或细胞内必定存在其相互作用蛋白。因此,筛选、鉴定OrfV F1L蛋白与宿主细胞互作的蛋白对解析病毒致病机制具有重要意义。本研究拟构建羔羊睾丸细胞酵母双杂交c DNA文库和OrfV F1L蛋白诱饵菌株,采用酵母双杂交技术筛选与OrfV F1L蛋白互作的宿主细胞蛋白,进一步采用回复杂交、免疫共沉淀（Co-IP）、His-Pull down对F1L蛋白与宿主细胞互作蛋白进行验证,激光共聚焦显微镜观察F1L蛋白与宿主细胞互作蛋白在细胞内的共定位。为探究OrfV的致病机制奠定基础。1.羔羊睾丸细胞酵母双杂交c DNA文库的构建及鉴定该试验旨在构建羔羊睾丸细胞酵母双杂交c DNA文库,并对其文库质量进行评价。采集羔羊睾丸制备LT细胞,稳定传代后利用TRIzol法提取LT细胞总RNA,对总RNA的浓度及纯度进行检测,运用SMART技术合成全长c DNA,通过同源重组的方法将p GADT7-Rec载体与c DNA共转化至Y187酵母感受态细胞,经营养缺陷型选择性培养基（SD/-Leu）筛选阳性克隆以构建c DNA文库,并对文库滴度、总容量、重组率及插入片段进行检测。结果显示,成功制备LT细胞,构建了LT细胞酵母双杂交c DNA文库,经检测文库滴度为2.33×10⁸CFU·m L^-1,文库总容量达3.5×10⁹CFU,文库重组率为100%,插入片段长度主要集中在250～2 000 bp。结果表明,构建的LT细胞c DNA文库质量良好,可用于后续酵母双杂交筛选。2.羊口疮病毒F1L蛋白诱饵菌株的构建及鉴定该试验旨在构建OrfV F1L蛋白诱饵菌株。提取Orf病料DNA,PCR扩增F1L基因,将其克隆至p MD18-T载体,经PCR、双酶切以及测序鉴定,获得p MD18-T-F1L克隆质粒;将p MD18-T-F1L克隆质粒和p GBKT7载体同时经限制性内切酶Eco RⅠ和NcoⅠ酶切后,连接转化至DH5α感受态细胞以构建诱饵质粒p GBKT7-F1L;采用PEG/Li Ac方法将p GBKT7-F1L诱饵质粒转化至Y2H Gold酵母感受态细胞,通过Western-Blot鉴定诱饵蛋白在酵母细胞中是否正确表达,再经SD/-Trp/-Leu（DDO）、SD/-Trp/-Leu/-His/-Ade（QDO）选择性培养基检测其自激活活性,同时测定诱饵菌株与空载体菌株在不同时间段的OD_{600 nm},对比两者生长曲线差异以检测诱饵蛋白是否有细胞毒性。结果显示,扩增出F1L基因大小为1 029 bp,成功构建p GBKT7-F1L诱饵质粒,及p GBKT7-F1L/Y2H Gold诱饵菌株。经Western-Blot验证表达的诱饵蛋白大小约为61 k Da,同时经DDO、QDO选择性培养基检测诱饵蛋白无自激活活性,OD_{600 nm}测定诱饵菌株生长曲线显示诱饵蛋白无毒性作用。结果表明,本试验成功构建OrfV F1L蛋白诱饵菌株,满足后续双杂交筛选试验条件。3.羊口疮病毒F1L蛋白与宿主细胞互作蛋白的筛选及鉴定该试验旨在筛选及鉴定在LT细胞上与OrfV F1L蛋白互作的细胞蛋白。将LT细胞c DNA文库与p GBKT7-F1L/Y2H Gold诱饵菌株进行杂交混合培养,经QDO和SD/-Trp/-Leu/-His/-Ade/X-α-gal/Ab A（QDO/X/A）选择性培养基进行3次筛选,再经回复杂交、Co-IP、His-Pull down以及激光共聚焦进行互作验证。结果显示,经酵母双杂交初步筛选有57个宿主细胞蛋白与F1L蛋白相互作用;进一步通过回复杂交试验,验证与F1L蛋白互作的宿主细胞蛋白有5个,分别是:真核翻译起始复合物3i（Eukaryotic translation initiation factor 3 subunit I,e IF3i）、α-辅肌动蛋白4（Alpha actinin 4,ACTN4）、纤维连接蛋白1（Fibronectin 1,FN1）、基质金属蛋白酶2（Matrix metallopeptidase 2,MMP2）和半乳糖凝集素1（Lectin galactoside-binding soluble 1,LGALS1）;从5个蛋白中选择LGALS1与F1L蛋白进行Co-IP和His-Pull down试验,均证实LGALS1蛋白与F1L蛋白在细胞内、外存在相互作用;利用激光共聚焦显微镜观察F1L蛋白与LGALS1蛋白共定位于细胞质。结果表明,OrfV的F1L蛋白与LT细胞中的LGALS1存在相互作用。更多还原

【Abstract】 Orf is a contact and epithelial zoonotic infectious disease caused by OrfVirus（OrfV）.OrfV mainly infects sheep and goats,and the incidence of lambs is as high as90%～100%.The main characteristic lesions are proliferative damage in lips and nose,which is an important infectious disease endangering the sheep industry.OrfV is a member of the genus Parapoxvirus,family Poxviridae and is an enveloped linear double-stranded DNA virus.F1L protein is a surface envelope protein of virus.It has been found that F1L protein has heparin binding activity,which is related to virus adsorption and invasion of host cells and plays an important role in the early stage of virus infection.Lamb testicular（LT）cells are sensitive cells for OrfV proliferation and there must be interacting proteins on the surface or inside of the cell membrane.Therefore,the screening and identification of OrfV F1L proteins interacting with host cells are of great significance for the analysis of viral pathogenesis.In this study,the yeast two-hybrid c DNA library of lamb testicular cells and OrfV F1L protein bait strain were constructed.The host cell proteins interacting with OrfV F1L protein were screened by yeast two-hybrid technology,and further verified by reverse hybridization,Co-IP and His-Pull down,colocalization of F1L protein interacting with host cells protein by confocal laser scanning microscopy.Lay the foundation for exploring the pathogenic mechanism of OrfV.1.Construction and identification of yeast two-hybrid c DNA library of lamb testicular cellsThe aim of this study was to construct a yeast two-hybrid c DNA library of lamb testicular cells and evaluate the quality of the library.The lamb testicular（LT）was collected to prepare LT cells.After stable passage,the total RNA of LT cells was extracted by TRIzol method,and the concentration and purity of total RNA were detected.The full-length c DNA was synthesized by SMART technology,and the p GADT7-Rec vector and c DNA were co-transformed into Y187 yeast competent cells by homologous recombination.Positive clones were screened by nutrient-deficient selective medium（SD/-Leu）to construct c DNA libraries,and the library titer,total capacity,recombination rate and insert were tested.The results showed that LT cells were successfully prepared and the yeast two-hybrid c DNA library of LT cells was constructed.The library titer was 2.33×10⁸ CFU·m L^-1,the total capacity of the library was 3.5×10⁹ CFU,the library recombination rate was 100%,and the length of the inserted fragment was mainly concentrated in 250-2 000 bp.The results indicated that the constructed c DNA library of LT cells was of good quality and could be used for subsequent yeast two-hybrid screening.2.Construction and identification of OrfV F1L bait strainThis experiment aimed to construct OrfV F1L protein bait strain.The DNA of Orf disease material was extracted,the F1L gene was amplified by PCR,and it was cloned into p MD18-T vector.After identification by PCR,double restriction digestion and sequencing to obtain p MD18-T-F1L clone plasmid.p MD18-T-F1L cloning plasmid and p GBKT7 vector were digested with restriction endonucleases Eco R I and Nco I at the same time,ligated and transformed into DH5αcompetent cells to construct bait plasmid p GBKT7-F1L.Transformation of p GBKT7-F1L decoy plasmid into Y2H Gold yeast competent cells using PEG/Li Ac method.Western-Blot was used to identify whether the bait protein was correctly expressed in yeast cells,and then SD/-Trp/-Leu（DDO）,SD/-Trp/-Leu/-His/-Ade（QDO）selective medium was used to detect its self-activation activity,and at the same time,the OD_{600 nm} of the bait strain and the empty vector strain at different time periods was determined,and the difference in the growth curves of the two strains was compared to detect whether the bait protein had cytotoxicity.The results showed that the amplified F1L gene was 1 029 bp,and the p GBKT7-F1L bait plasmid and the bait strain p GBKT7-F1L/Y2H Gold were successfully constructed.And the size of the bait protein verified by Western-Blot was about 61 k Da.At the same time,the bait protein had no self-activation activity detected by DDO and QDO selective media.The growth curve of the bait strain determined by OD_{600 nm} showed that the bait protein had no toxic effect.The results indicated that the OrfV F1L protein bait strain was successfully constructed in this experiment,which met the conditions of subsequent two-hybrid screening.3.Screening and identification of OrfV F1L protein and host cell interaction proteinThis assay aims to screen and identify cellular proteins that interact with OrfV F1L protein on LT cells.Hybridization of LT cell c DNA library with p GBKT7-F1L/Y2H Gold bait strain.Screening 3 times on selective medium of QDO and SD/-Trp/-Leu/-His/-Ade/X-α-gal/Ab A（QDO/X/A）,and then verified by revertant hybridization,Co-IP,His-Pull down and laser confocal for interactions.The results showed that 57 host cell proteins preliminarily screened by yeast two-hybrid system interacted with F1L protein.Furthermore,five host cell proteins interacting with F1L protein were identified by revertant hybridization,namely eukaryotic translation initiation factor 3 subunit I（e IF3i）,alpha actinin 4（ACTN4）,fibronectin 1（FN1）,matrix metallopeptidase 2（MMP2）and LGALS1.LGALS1 and F1L proteins were selected from the five proteins for Co-IP and His-Pull down tests,which confirmed that LGALS1 protein and F1L protein interacted in and out of cells.Colocalization of F1L protein and LGALS1protein in cytoplasm by laser confocal for interactions,and colocalization of F1L protein and LGALS1 protein in cytoplasm by confocal laser scanning microscopy.The results indicated that F1L protein of OrfV interacted with LGALS1 in LT cells.更多还原