文档介绍：人核心蛋白聚糖的克隆及其在大肠杆菌中的表达
作者:王利民温铭杰李慎涛

【摘要】目的在大肠杆菌中高效表达人核心蛋白聚糖,并建立适当的纯化方法,制备重组蛋白。方法根据GenBank中人核心蛋白聚糖的序列设计特异性引物,从人肝cDNA文库中PCR扩增核心蛋白聚糖的编码序列,克隆到原核表达载体pET42a中,转化大肠杆菌BL21(DE3),经IPTG诱导表达后,获得以高效表达的目的重组蛋白;用SDSPAGE和质谱鉴定重组蛋白。结果重组蛋白主要以包涵体形式表达,SDSPAGE显示其分子质量与预期结果相符;经质谱鉴定,表达的重组蛋白为人核心蛋白聚糖。结论成功地在大肠杆菌中高效表达了人核心蛋白聚糖,并纯化得到人核心蛋白聚糖蛋白,经鉴定,纯化所得到的蛋白为人核心蛋白聚糖。
【关键词】核心蛋白聚糖;原核表达;蛋白纯化;质谱鉴定
【Abstract】 Objective To express the human decorin in Escherichia coli effectively, to set up a appropriate protocol for purification of the binant protein and to prepare the expressed human Specific primers for human decorin were designed according to its sequence published in GenBank. DNA encoding the human decorin was PCR amplified from a human liver cDNA library, and was cloned into pET42, obtaining the plasmid of DCN
pET42a. The plasmid was transformed into BL21(DE3) strain of E. induction with IPTG, the human decorin was effectively expressed in Human decorin was expressed in in the form of inclusion body. Identifications by SDSPAGE and mass spectrometry demonstrated that the binant protein was human Human decorin was essfully expressed in E. suitable protocol for purification of the binant protein was set up. After purification, we got pure protein of the expressed human decorin.
【Key words】 decorin,prokaryotic expression,purif