知识与财富的链接
以高效原核表达载体 pBV220为骨架载体,应用单链寡核苷酸引物插入法在pBV220多克隆位点的下游插入了六聚组氨酸融合标签编码序列(6×His-Tag)、羟胺和凝血酶蛋白切割位点, 并增加了Xho I和Kpn I酶切位点和强终止密码子TAA, 将此新质粒命名为 pBV223。以此载体表达的目的蛋白在羧基端(C端)带有六聚组氨酸尾以利于通过固定化金属亲和层析快速纯化目的蛋白, 酶切及核苷酸序列分析验证了我们的设计。将终止密码缺失突变的nm23-H1 cDNA克隆入pBV223载体中, 在大肠杆菌DH5a中成功地表达了Nm23-H1蛋白, 通过镍(Ni)亲和层析一步即简单、快速地得到了纯化蛋白。我们所应用的单链寡核苷酸引物插入直接进行定向克隆的方法是目前为止最简便的方法。
A single-stranded oligonucleotides containing a 6 histidine sequence, a hydroxylamine cleavage site, a thrombin cleavage site, and stop codon TAA were inserted into the polylinker's downstream of prokaryotic expression vector pBV220 between BamHI and PstI. The resultant vector is named pBV223. Proteins expressed in this vector will have a 6 histidine tail as affinity handy fused to their C terminus and can be quickly purified by one step immobilized metal affinity chromatography (IMAC). This plasmid is verified by restriction map and DNA sequencing. Subsequently, the metastasis suppressor gene nm23-H1 cDNA (without the stop codon) was cloned into vector PBV223 in frame with the 6-histidine sequence, hydroxylamine and thrombin cleavage sites. The soluble nm23-H1 fusion protein was successfully induced in the bacterial DH5a and easily purified with Ni chromatograph. These results indicated that the strategy to clone the single-stranded oligonucleotides directly into the restriction sties between BamH I and Pst I in the pBV220 vector is the simplest and cost-effective method.
