摘要
为了构建原核表达载体pSBET-His。pSBETa质粒经XbaI和BamH I限制性内切酶双酶切,获得原核表达载体的骨架。以pET28a(+)为模板,通过高保真PCR法应用T7 promoter引物和T7 terminator引物对pET28a(+)载体T7表达区域进行扩增,PCR产物电泳纯化后经双酶切和纯化获得138 bp含His-Tag序列的片段。将pSBET原核表达载体骨架和含有His-Tag序列的片段进行连接后转入DH5α感受态细胞中,经菌落PCR及酶切筛选构建pSBET-His。成功构建了pSBET-His原核表达载体。应用该载体表达的蛋白质,可采用镍离子亲和层析法进行纯化。该研究为应用pSBET-His载体进行烟草丛顶病毒ORF4的原核表达研究奠定了基础。
The aim of the research was to construct prokaryotic expression vector pSBET-His, Double restriction-enzyme digestion was conducted on pSBE- Ta plasmid by restriction endonucleases Xba Ⅰ and Bam H Ⅰ to obtain the framework of the prokaryotic expression vector. With pET28a （ ＋ ） as template, T7 expression region of pET28a （ ＋ ） vector was amplified with T7 promoter primer and T7 terminator premier by high fidelity PCR. Double restriction-enzyme digestion and purification of PER products were conducted to obtain the fragment of 138 bp that contained His-Tag sequence after electrophoresis purification of WaR products. The framework of prokaryotic expression vector pSBET and the fragment that contained His-Tag sequence were ligated and transformed into DH5α competent cell, and pSBET-His was obtained through colony PCR and enzyme-digestion screening.Pmkaryotic expression vector was comtructed successfully. The proteins expressed by this vector could be purified by Ni ion affinity chromatography method, The research laid the fotmdation for studying pmkaryotic expression of tobacco bushy top virus ORF4 with pSBET-His vector.
出处
《安徽农业科学》
CAS
北大核心
2007年第27期8443-8443,8446,共2页
Journal of Anhui Agricultural Sciences
基金
云南省烟草公司科技项目(04A18)
