猪流行性腹泻病毒纳米抗体融合蛋白真核表达及鉴定  

Eukaryotic Expression and Identification of PEDV Nanobody Fusion Protein

作　　者：郭涛 李村院 刘开平 李晓悦 胡瑞瑞 李雅心 王小奎[1] 倪伟[1] 胡圣伟[1] GUO Tao;LI Cun-yuan;LIU Kai-ping;LI Xiao-yue;HU Rui-rui;LI Ya-xin;WANG Xiao-kui;NI Wei;HU Sheng-wei(College of Life Science,Shihezi University,Shihezi,Xinjiang,832000,China;College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang,832000,China)

机构地区：[1]石河子大学生命科学学院,新疆石河子832000 [2]石河子大学动物科技学院,新疆石河子832000

出　　处：《动物医学进展》2021年第4期17-21,共5页Progress In Veterinary Medicine

基　　金：2019年自治区研究生科研创新项目(XJ2019G11);“兵团英才”第二周期第一层次培养人选项目。

摘　　要：为防控猪流行性腹泻病毒(PEDV)病的流行,构建了靶向PEDV NB3-NB6-Fc纳米抗体融合蛋白的真核表达载体,并探究能否在毕赤酵母中分泌表达。通过实验室保存的pUC57-NB3-NB6-Fc质粒,成功构建分泌表达载体pPIC9K-NB3-NB6-Fc,经SacⅠ酶将其线性化并电转化入毕赤酵母GS115菌株。经过MD、梯度G418-YPD平板和PCR方法筛选多拷贝阳性重组转化子。经鉴定为阳性重组菌株用8 mL/L甲醇在29℃条件下诱导72 h,用SDS-PAGE和Western blot方法检测上清液中重组蛋白的表达情况。结果显示,成功诱导出大小为53.2 ku的分泌型重组蛋白,经Swiss-model在线预测该蛋白具有相对紧密的空间结构。成功获得pPIC9K-NB3-NB6-Fc-GS115分泌表达菌株,可为PED的预防和治疗提供材料。In order to prevent porcine epidemic diarrhea virus(PEDV),the eukaryotic expression vector of NB3-NB6-Fc nanobody fusion protein targeting PEDV was constructed,and whether it could be secreted and expressed in Pichia pastoris was explored.The secretion expression vector pPIC9K-NB3-NB6-Fc was successfully constructed through the saved pUC57-NB3-NB6-Fc plasmid,then it was linearized with SacⅠenzyme and electroporated into Pichia pastoris GS115 strain.Multiple copies of positive recombinant transformants were screened by MD,gradient G418-YPD plate and PCR method.The recombinant strains identified as positive were induced with 8 mL/L methanol at 29℃for 72 h,and the expression of recombinant protein in the supernatant was detected by SDS-PAGE and Western blot methods.The results showed that a secreted recombinant protein with a size of 53.2 ku was successfully induced.The Swiss-model online predicted that the protein has a relatively tight spatial structure.The secretion expression strain pPIC9K-NB3-NB6-Fc-GS115 provided new ideas for the prevention and treatment of PED in the future.

关 键 词：猪流行性腹泻病毒 纳米抗体 融合蛋白 毕赤酵母 分泌表达 

分 类 号：S852.65[农业科学—基础兽医学]