胞内劳森菌鞭毛素蛋白FliC与猪肠上皮细胞互作蛋白的研究  

The study of the Lawsonia intracellularis flagellin protein FliCinteracting proteins in porcine intestinal epithelial cells

作　　者：于振兴 张兰桥 杨莹莹 张锦华[1] 何后军[1] 戴益民[1] YU Zhen-xing;ZHANG Lan-qiao;YANG Ying-ying;ZHANG Jin-hua;HE Hou-jun;DAI Yi-min(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045,China;College of Biology Science and Engineering,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045 [2]江西农业大学生物科学与工程学院,江西南昌330045

出　　处：《中国预防兽医学报》2021年第12期1324-1329,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31660720)。

摘　　要：为鉴定胞内劳森菌鞭毛蛋白FliC与猪肠上皮细胞中的互作蛋白,本研究利用PCR方法从回肠炎疫苗提取的DNA中扩增胞内劳森菌鞭毛素基因LI0710,并将其克隆至原核表达载体pET32a(+)中构建重组质粒pET32aLI0710,经双酶切及测序鉴定正确后转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导并经SDS-PAGE检测。结果显示,获得可溶且与硫氧还蛋白-6×His-S标签(Trx-His-S)融合表达的重组FliC蛋白(Trx-His-S-FliC)。采用镍柱亲和层析法纯化Trx-His-S和Trx-His-S-FliC,并分别与猪肠上皮细胞(IPEC-J2)裂解液孵育后,利用anti-His抗体进行免疫共沉淀试验(Co-IP),并对沉淀物利用液质联用串联质谱(LC/MS/MS)技术分析与FliC互作的宿主蛋白,利用Proteome Discoverer 1.4软件在UniProtKB/Swiss-Prot数据库中对互作的宿主蛋白进行肽指纹图谱分析。Co-IP结果显示,检测到了多个与Trx-His-S-FliC互作的宿主蛋白,且通过LC/MS/MS技术共鉴定到50个与FliC互作的蛋白。这些互作蛋白包括蛋白酶、多个核糖体蛋白和线粒体蛋白,广泛参与了细胞质糖酵解、氨基酸代谢、ATP跨膜转运等功能。为了证明质谱数据的可靠性,选择与FliC互作最强的胰蛋白酶经western blot验证,结果显示,胰蛋白酶与FliC蛋白确实存在互作,表明质谱数据可靠。本研究首次鉴定到多个与胞内劳森菌鞭毛蛋白FliC互作的宿主蛋白,为研究FliC蛋白的功能奠定了基础。To identify the interacting protein of Lawsonia intracellularis flagellin FliC in porcine intestinal epithelial cells,the flagellin gene LI0710 was amplified from the extracted DNA of L.intracellularis vaccine by PCR and then inserted into the prokaryotic expression vector pET32 a(+)to construct the recombinant plasmid pET32 a-LI0710.After verification by enzyme digestion and sequencing,the plasmid pET32 a-LI0710 was transformed into Escherichia coli BL21(DE3),the expression of flagellin FliC was induced with IPTG and analyzed by SDS-PAGE.The results showed that the soluble and thioredoxin-His-S tagged FliC(Trx-His-S-FliC)was obtained.The Trx-His-S and Trx-His-Flic were purified by Ni-NTA agarose resin,incubated with porcine intestinal epithelial(IPEC-J2)cell lysate,respectively,and Co-immunoprecipitated(Co-IP)by anti-His antibody.The precipitates were detected by liquid combine tandem mass spectrometry(LC/MS/MS)and the peptide fingerprints of related proteins were analyzed in UniProtKB/Swiss-Prot database using Proteome Discoverer 1.4 software.The results of Co-IP showed that many host proteins which interacted with Trx-His-S-FliC had been detected and a total of 50 FliC-interacting proteins were identified.These FliC-interacting proteins mainly include proteases,ribosomal and mitochondrial proteins and involved extensively in cytoplasmic glycolysis,amino acid metabolism,atp transmembrane transport.Aiming to prove the reliability of the mass spectrometry data,the trypsin which was predicted to has the strongest interactivity with FliC was selected for the interaction which was verified by western blot.The results showed that trypsin interacted with FliC protein,indicating the reliability of mass spectrometry data.In this study,several host proteins interacting with L.intracellularis flagellin FliC were identified for the first time,laying a foundation for further research on the function of FliC protein.

关 键 词：增生性肠炎 胞内劳森菌 鞭毛素蛋白FliC 免疫共沉淀 病原菌-宿主相互作用 

分 类 号：S852.61[农业科学—基础兽医学]