重组小反刍兽疫病毒血凝素蛋白纯化体系的优化  

Optimization of the purification system of recombinant hemagglutininprotein from peste des petits ruminants virus

作　　者：胡林杰 朱学亮[1] 彭泓蛟 邓瑞雪 潘春容 孙跃峰[1] 蒙学莲[1] HU Linjie;ZHU Xueliang;PENG Hongjiao;DENG Ruixue;PAN Chunrong;SUN Yuefeng;MENG Xuelian(State Key Laboratory for Animal Disease Control and Prevention/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2024年第1期79-86,共8页Chinese Veterinary Science

基　　金：边境地区和重要口岸动物病原监测溯源技术研究项目(2021YFD1800500);兰州市人才创新创业项目(2022-RC-22)。

摘　　要：本试验利用发酵技术诱导表达了小反刍兽疫病毒H蛋白胞外区(PPRV tH),优化建立了一套纯化重组PPRV tH的完整工艺体系。通过发酵表达获得的10 L菌液,菌体湿重约达30 g/L,均质机破碎菌体获得包涵体;Tris-0缓冲液(pH=8.0)洗涤包涵体3次,每1 g包涵体加入5 mL含20 mmol/L咪唑的Tris-E缓冲液(pH=8.0)重悬,4℃温和旋转溶解过夜,离心收集上清;按体积比1∶2将层析填料Chelaing Sepharose Fast Flow与上清混匀,以流速1~2 mL/min加入层析柱;依次通过10倍柱体积含50、100、400和500 mmol/L咪唑的Tris-E缓冲液(pH=8.0),4℃静置2 h进行洗脱。优化后的纯化体系可得到较纯的重组PPRV tH蛋白,利用灰度分析可知纯化后的重组PPRV tH蛋白纯度能达到85%。本试验提供的纯化体系可快速、简便、低成本、大批量纯化重组PPRV tH蛋白,而且纯化获得的PPRV tH蛋白具有良好的反应原性。In this experiment,the extracellular domain of hemagglutinin of peste des petits ruminants virus(PPRV tH)was expressed by fermentation technology,and a complete purification system for PPRV tH was established by optimization.The cell wet weight was about 30 g per liter of culture broth by fermentation.The inclusion body was obtained by homogenizer and washed three times with Tris-0 buffer(pH=8.0).Then per 1 g of inclusion bodies was resuspended with 5 mL Tris-E buffer(pH=8.0)containing 20 mmol/L imidazole,and dissolved overnight at 4℃by gentle rotation.The supernatant was retained by centrifugation.The Chelaing Sepharose Fast Flow was mixed with the supernatant at a volume ratio of 1∶2 and added to the column at a flow rate of 1—2 mL/min.The recombinant PPRV tH was eluted by passing through 10 times the column volume of Tris-E buffer(pH=8.0)with a stepwise gradient of imidazole(50,100,400 and 500 mmol/L)for 2 h at 4℃.The purity of purified recombinant PPRV tH protein using optimized purification system can reach 85%by gray scale analysis.Its fast,simple,low cost and higher final purity properties make it excellent for purifing the recombinant PPRV tH in this experiment and the purified PPRV tH protein has good reactogenicity.

关 键 词：小反刍兽疫病毒 血凝素蛋白 蛋白纯化 优化 

分 类 号：S852.659.5[农业科学—基础兽医学]