猪流行性腹泻病毒COE蛋白在TP309水稻细胞中的表达及纯化  

Expression and Purification of Porcine Epidemic Diarrhea Virus COE Protein in TP309 Rice Cells

作　　者：陈金炫 史倩倩 潘世源 陈恒耀 寇少康 楚红燕 张磊[1] 张二芹 张改平[1,2] CHEN Jin-xuan;SHI Qian-qian;PAN Shi-yuan;CHEN Heng-yao;KOU Shao-kang;CHU Hong-yan;ZHANG Lei;ZHANG Er-qin;ZHANG Gai-ping(International Joint Research Center of National Animal Immunology,College of Veterinary Medicine,Henan Agricultural University,Zhengzhou,Henan 450046,China;Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou,Henan 450002,China)

机构地区：[1]河南农业大学动物医学院,国家动物免疫学国际联合研究中心,河南郑州450046 [2]河南省农业科学院动物免疫学重点实验室,河南郑州450002

出　　处：《动物医学进展》2025年第4期1-5,共5页Progress In Veterinary Medicine

基　　金：河南省高等学校重点科研项目(25A230003);河南省重大科技专项(221100110600)。

摘　　要：为获得TP309水稻细胞表达的猪流行性腹泻病毒(PEDV)COE蛋白,根据水稻密码子偏爱性优化并合成COE基因,构建植物重组质粒pCAMBIA1300-COE,通过根癌农杆菌EHA105介导法将pCAMBIA1300-COE转入水稻愈伤组织。经过暗培养和潮霉素B筛选新愈伤;PCR鉴定阳性愈伤;阳性愈伤研磨后制成细胞并进行培养,阳性细胞经无糖诱导培养10 d后,Western blot鉴定表达COE蛋白;并利用Q阴离子层析和MMC阳离子层析纯化COE蛋白。结果显示,经潮霉素筛选获得73个新愈伤,PCR检测有50个阳性愈伤,阳性率约为68.49%,Western blot检测有15个水稻细胞株表达COE蛋白,COE蛋白经Q和MMC纯化后,其纯度达到50%左右。这些研究结果对制备猪流行性腹泻亚单位疫苗具有重要意义。In order to obtain porcine epidemic diarrhea virus COE protein expressed from TP309 rice cells,COE gene was optimized and synthesized according to rice codon preference.The plant recombinant plasmid pCAMBIA1300-COE was constructed and transferred into rice callus by Agrobacterium tumefaciens EHA105.The new callus was screened by dark culture and hygromycin B.PCR identified positive callus.The positive callus was ground into cells and cultured.The positive cells were cultured without sugar for 10 days and the expression protein was identified by Western blot.COE protein was purified by Q anion chromatography and MMC cation chromatography.The results showed that 73 new callus were obtained by hygromycin screening,50 positive callus were detected by PCR.The positive rate was 68.49%.There were 15 rice cell lines expressing COE protein by Western blot.The purity of COE protein was about 50%after purification by Q and MMC.Therefore,these conclusions are of great significance for the preparation of subunit vaccines.

关 键 词：猪流行性腹泻 COE蛋白 水稻愈伤 水稻植株 纯化 

分 类 号：S852.651[农业科学—基础兽医学] S858.28[农业科学—兽医学]