一种新穿膜肽的构建、表达及其活性检测

通过对穿膜肽TAT-PTD构效关系的分析和结构改造，构建能有效进入血脑屏障且低毒的新穿膜肽。该研究用分子克隆技术构建出表达型载体pET28a—T1-GFP，重组质粒在大肠杆菌Ecoli BL21（DE3）中表达融合蛋白His-T1-GFP，经组氨酸亲和层析纯化后，用荧光显微镜和免疫组化的方法来检测His—T1-GFP在活体动物小白鼠体内的跨膜递送作用。经测序证实成功构建了表达型载体pET28a—T1-GFP，融合蛋白His—T1-GFP在大肠杆菌EcoliB L21（DE3）中表达率为20％。经组氨酸亲和纯化，得到纯度达85％的融合蛋白。SDS-PAGE和Western Blotting显示纯化蛋白为His-T1-GFP，动物实验表明融合蛋白His—T1-GFP能够有效跨过血脑屏障，主要分布在大脑皮层和海马回。该研究成功地构建了-种新的穿膜肽-T1，为其携带有生物活性的大分子物质到达脑部进行蛋白治疗奠定了基础。

Construction, Expression and Activity Assay of A Novel Cell Penetrating Peptide

To construct a novel cell penetrating peptide(CPP) that can effectively permeate into blood brain barrier(BBB) with few side effects in vivo via structure-function relationship analysis and structural modification of the penetrating peptide.TAT-PTD,recombinant expression plasmid pET28aT1-GFP was constructed by DNA clone.The expression of His-T1-GFP in E.coli BL21(DE3) transformed with recombinant expression plasmid pET28a-T1-GFP was induced by IPTG and the expressed protein was purified by Ni-NTA resin chromatography.By using the fluorescence microscope and immunohistochemical method the permeation efficacy of His-T1-GFP was also studied in vivo.The recombinant vector pET28a-T1-GFP was successfully constructed.And the expression level of His-T1-GFP in E.coli BL21(DE3)/pET28a-T1-GFP was up to 20% of the total cellular protein.The purity of His-T1-GFP obtained after the Ni-NTA resin chromatography purification was 85%.The fusion protein His-T1-GFP was successfully expressed via SDS-PAGE and Western Blotting analysis.Finally,His-T1-GFP can effectively penetrate across the BBB in mice,which was mainly distributed in hippocamp and cerebral cortex.In conclusion,a novel cell penetrating peptide(CPP) that can effectively permeate into BBB in mice was successfully constructed.