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目的 获取人His-AWP1融合蛋白,为下阶段深入研究AWP1的结构、功能及筛取与其相互作用的蛋白打下基础 方法 应用逆转录聚合酶链反府(RT-PCR)法从人ECV304内皮细胞系中克隆AWP1 cDNA,并将其重组于能表达6个组氨酸残基的原核表达质粒DET-14b中。经酶节、序列鉴定,选择正确重组克隆,将其质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,用Ni2+-NTA His柱纯化和SDS-PAGE分离蛋门。结果克隆到一个627 bp的AWP1 cDNA片段.重组质粒目的DNA测序正确.纯化出了一个分子量约为38 kD的融合蛋白。结论用基因工程方法在原核细胞表达并成功纯化出His-AWP融合蛋白。
OBJECTIVE To obtain the His-AWP1 fusion protein and lay the foundation for the further study of the structure, function and screening of proteins interacting with AWP1 in the next stage. The reverse transcription polymerase chain reaction (RT-PCR) method was used to detect the expression of AWP1 from human ECV304 endothelial cell line The AWP1 cDNA was cloned and recombined into the prokaryotic expression plasmid DET-14b capable of expressing six histidine residues. The correct clones were selected by enzyme digestion and sequence identification. The plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The eggs were isolated by Ni2 + -NTA His column and SDS-PAGE. As a result, a 627 bp cDNA fragment of AWP1 was cloned, and a DNA fragment of 38 kD was purified. Conclusion The His-AWP fusion protein was successfully expressed in prokaryotic cells by genetic engineering.