马铃薯Y病毒衣壳蛋白的表达、纯化与结晶条件筛选

Expression and purification of potato Y virus coat protein and screening of crystallization conditions

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Eco R 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;PVY CP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。

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 Eco R 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.