纯化马铃薯S病毒原核表达重组CP的简便方法

A Simple Method for Purification of Prokaryotic Expression-derived Recombinant CP of Potato Virus S

用PVS-CP基因的原核表达载体pBAD-PVS转化受体菌TOP10获得了重组菌TOP10(pBAD-PVS),经阿拉伯糖诱导该重组菌表达出了46kDa的特异性融合蛋白,在Western blotting图谱上该蛋白与PVS特异性抗体(IgG)反应,呈现一条阳性杂交带,表明46kDa蛋白带是PVS-CP基因正确表达的产物。从重组菌中提取包涵体,包涵体经过4mol/L尿素洗涤后用超纯水4℃过夜溶解,所获溶液即为高纯度PVS重组CP水溶液,也就是说本研究建立了一种获得高纯度重组CP的非常简便的方法。

The prokaryotic expression vector of CP gene of potato virus S (pBAD -PVS) was introduced into E .coli strain TOP10 ,then the recombinant strain TOP10 (pBAD-PVS) was induced with L -arabi-nose ,and a 46kDa specific protein band was found in SDS pattern of total protein .In Western blotting a-nalysis ,the 46kDa protein reacted with PVS -IgG ,and a positive band was observed ,the result indicated that the 46kDa protein was the correct expression product of PVS -CP gene .Inclusion body was extracted from TOP10 (pBAD -PVS) and washed with 4 mol/L urea .The washed inclusion body was suspended in ultra pure water and kept at 4℃ overnight .The resulting solution was indeed the high -purity recombi-nant CP ,a very simple purification method of PVS recombinant CP was established .