托高土病毒核蛋白的异源表达、纯化与结晶  

Heterologous Expression,Purification and Crystallization of Thogoto Virus Nucleoprotein

作　　者：马林 鞠金鑫 李琳 秦晓春 董士尚 MA Lin;JU Jinxin;LI Lin;QIN Xiaochun;DONG Shishang(School of Biological Science and Technology,University of Jinan,Jinan 250022,Shandong,China)

机构地区：[1]济南大学生物科学与技术学院,山东济南250022

出　　处：《济南大学学报（自然科学版）》2024年第6期707-712,共6页Journal of University of Jinan(Science and Technology)

基　　金：山东省自然科学基金项目(ZR2020QC057);泰山学者青年专家计划项目(tsqn201812079)。

摘　　要：为了获得托高土病毒核蛋白氮端结构域的蛋白(THOV-NP_(N))晶体,进一步探究其结构和功能,依次采用镍亲和层析、肝素亲和层析方法纯化大肠杆菌异源表达获得的THOV-NP_(N),利用凝胶过滤层析及十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法检测THOV-NP_(N)的均一性和纯度,分别用坐滴法和悬滴法筛选和优化THOV-NP_(N)结晶条件,采用质谱法验证所得的晶体。结果表明:所使用的提取方法可以获得纯度较高、性质均一的THOV-NP_(N),在培养温度16℃,THOV-NP_(N)的质量浓度为25.0 g/L,池液中磷酸氢二铵、柠檬酸钠、氯化钠的浓度分别为0.8、0.1、0.2 mol/L及pH=5.0的条件下可以获得粒径为0.05 mm、形状近似正方体的THOV-NP_(N)晶体。To obtain crystals of the N-terminal domain of the nucleoprotein of Thogoto virus(THOV-NP_(N))for further exploration of their structure and function,nickel affinity chromatography and heparin affinity chromatography methods were sequentially used to purify THOV-NP_(N)expressed heterologously in Escherichia coli.Gel filtration chromatography and sodium dodecyl sulfonate-polyacrylamide gel electrophoresis were employed to assess the homogeneity and purity of THOV-NP_(N).Crystallization conditions for THOV-NP_(N)were screened and optimized using both sitting-drop and hanging-drop methods,and the obtained crystals were validated using mass spectrometry.The results show that the methods employed enabled the purification of high-purity and homogeneous THOV-NP_(N).Under the conditions of cultivation temperature of 16℃,mass concentration of 25.0 g/L of THOV-NP_(N),and concentration of 0.8,0.1,and 0.2 mol/L for ammonium dihydrogen phosphate,sodium citrate,and sodium chloride in the pool liquid respectively,with pH of 5.0,THOV-NP_(N)crystals with particle size of 0.05 mm and a shape approximating can be obtained.

关 键 词：托高土病毒 核蛋白 异源表达 纯化 结晶 

分 类 号：Q71[生物学—分子生物学]