PTD-Lmp-3融合蛋白的纯化、表达和鉴定

目的构建融合蛋白PTD-Lmp-3的表达质粒,并进行诱导表达、纯化及鉴定。方法利用基因重组技术构建pET43.1a-PTD-Lmp-3融合蛋白表达载体,并在大肠埃希菌BL21（DE3）中表达融合蛋白,经Ni2＋-NTA层析纯化,SDS-PAGE和Western blot鉴定目的蛋白。结果成功构建了表达质粒pET43.1a-PTD-Lmp-3,表达的融合蛋白经SDS-PAGE分析,经IPTG诱导表达的总菌体蛋白在融合蛋白的相应位置PTD-LMP-3约为22 kD,可溶性分析发现融合蛋白主要以上清形式存在,纯化后得到了目的蛋白。Western blot鉴定显示表达蛋白具有抗原性。结论成功构建了PTD-Lmp-3的重组表达载体,使融合蛋白在大肠埃希菌BL21（DE3）中高效表达,亲和层析后获得了纯化目的蛋白,为进一步研究奠定了基础。

Purification, Expression and Verification of PTD-Lmp-3 Fusion Protein

Objective To clone,express,purify the LIM mineralization protein containing protein transduction domain of HIV-1 Trans-Activator(TAT PTD-Lmp-3). Methods The sequence coding TAT PTD-Lmp-3 fusion gene was amplified by PCR and cloned into the expression plasmid pET43.1a.After sequence analysis,the vector containing the fusion gene was transduced into the E.coli.BL21(DE3)plysS,which was induced with IPTG to express the TAT PTD-Lmp-3 fusion proteins.Ni2 -NTA resin was used to purify the products,which were verified by means of SDS-PAGE and Western blot analysis subsequently.Results The TAT PTD-Lmp-3 expression vector was successfully constructed.The fusion protein TAT PTD-Lmp-3 was about 22kD,which could react immunologically with anti-his-tag monoclonal antibody.Conclusions The TAT PTD-Lmp-3 expression vector is successfully constructed,and efficient expression of PTD-Lmp-3 fusion proteins the E.coli and purification of interest protein lay a basis for further study.