马铃薯Y病毒衣壳蛋白的表达、纯化与结晶条件筛选 |
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引用本文:蒋君梅,杜巧丽,陈美晴,严云龙,谢鑫,李向阳.马铃薯Y病毒衣壳蛋白的表达、纯化与结晶条件筛选.植物保护学报,2022,49(2):508-514 |
DOI:10.13802/j.cnki.zwbhxb.2022.2020170 |
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作者 | 单位 | E-mail | 蒋君梅 | 贵州大学, 绿色农药与农业生物工程教育部重点实验室, 贵州大学精细化工研究开发中心, 贵阳 550025 | | 杜巧丽 | 贵州大学农学院, 农业微生物特色重点实验室, 贵阳 550025 | | 陈美晴 | 贵州大学农学院, 农业微生物特色重点实验室, 贵阳 550025 | | 严云龙 | 贵州大学, 绿色农药与农业生物工程教育部重点实验室, 贵州大学精细化工研究开发中心, 贵阳 550025 贵州大学农学院, 农业微生物特色重点实验室, 贵阳 550025 | | 谢鑫 | 贵州大学农学院, 农业微生物特色重点实验室, 贵阳 550025 | xiexin2097757@163.com | 李向阳 | 贵州大学, 绿色农药与农业生物工程教育部重点实验室, 贵州大学精细化工研究开发中心, 贵阳 550025 | xyli1@gzu.edu.cn |
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中文摘要:为研究马铃薯Y病毒(potato virus Y,PVY)衣壳蛋白(coat protein,CP)在体外表达及CP重组蛋白的晶体生长条件,通过EcoR I/Hind III酶切及连接技术构建pET-32a-PVY CP原核表达载体,对PVY CP的诱导剂浓度进行优化,利用蛋白质纯化及脱盐技术对PVY CP进行纯化和脱盐,并分析PVY CP重组蛋白的晶体生长条件。结果表明,成功构建了pET-32a-PVY CP原核表达载体; PVYCP在大肠杆菌Escherichia coli感受态细胞BL21 (DE3)原核表达系统中,于16℃下利用0.8 mmol/L异丙基-β-D-硫代吡喃半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达可获得高浓度的PVY CP可溶性蛋白; PVY CP重组蛋白在二水甲酸镁处理下可生长出棒状结构晶体。 |
中文关键词:马铃薯Y病毒 衣壳蛋白 可溶性蛋白 表达纯化 结晶 |
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Expression and purification of potato Y virus coat protein and screening of crystallization conditions |
Author Name | Affiliation | E-mail | JIANG Jun-mei | Fine Chemical Research and Development Center | | DU Qiao-li | Key Laboratory of Agricultural Microbiology, College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China | | CHEN Mei-qing | Key Laboratory of Agricultural Microbiology, College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China | | YAN Yun-long | Fine Chemical Research and Development Center Key Laboratory of Agricultural Microbiology, College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China | | XIE Xin | Key Laboratory of Agricultural Microbiology, College of Agriculture, Guizhou University, Guiyang 550025, Guizhou Province, China | xiexin2097757@163.com | LI Xiang-yang | Fine Chemical Research and Development Center | xyli1@gzu.edu.cn |
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Abstract:In order to study the in vitro expression of potato virus Y (PVY) coat protein (CP) and the crystal growth conditions of PVY CP recombinant protein, pET-32a-PVY CP prokaryotic expression vector was constructed using EcoR I/Hind III digestionand linking technology, constructing, the concentration of inducer IPTG PVY CP was optimized forthe prokaryotic expression system, PVY CP was expressed and purified in large quantities using protein purification and desalting technology, and the crystal growth conditions of PVY CP recombinant protein were analyzed. The results showed that the pET- 32a-PVY CP prokaryotic expression vector was successfully constructed. The PVY CP was obtained in the Escherichia coli BL21 (DE3) prokaryotic expression system at 16℃, and 0.8 mmol/L IPTG could induce expression to obtain high purity PVY CP. The PVY CP recombinant protein grew rod-like crystals under the conditionsin presence of magnesium formatedihydrate.
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keywords:potato virus Y coat protein(CP) soluble protein expression purification crystallization |
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