TAT/LMP-3融合蛋白的制备及其诱导成骨活性的初步研究  

Preparation of TAT/LMP-3 fusion protein and a primary study of its osteogenic effects

作　　者：郑文杰[1] 罗刚[1] 向强[1] 李长青[1] 周跃[1] 

机构地区：[1]第三军医大学新桥医院骨科

出　　处：《第四军医大学学报》2009年第20期2093-2097,共5页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30700892);重庆市自然科学基金(CSTC;2007BB5058)

摘　　要：目的:表达并纯化具有入胞转导能力并保持骨诱导活性的重组融合蛋白TAT/LMP-3,观察融合蛋白TAT/LMP-3对人骨髓间充质干细胞的入胞转导活性和诱导其成骨分化的效能.方法:采用基因工程技术构建原核表达载体pET43.1a-TAT/LMP-3和pET43.1a-LMP-3并转化大肠杆菌BL21(DE3),IPTG诱导融合蛋白表达,Ni-NTA树脂亲和层析柱纯化后进行初步鉴定,同时制备重组蛋白LMP-3的多克隆抗体.将融合蛋白与人骨髓间充质干细胞共孵育,Western Blot技术分析融合蛋白的入胞效应,检测成骨细胞标志性基因表达以分析重组蛋白对人骨髓间充质干细胞成骨分化的影响.结果:成功地构建了pET43.1a-TAT-LMP-3融合基因原核表达载体,获得了TAT/LMP-3的可溶性表达,纯化后融合蛋白TAT/LMP-3纯度大于90%.成功制备了TAT/LMP-3的兔源性多克隆抗体.Western Blot分析证实TAT-LMP-3融合蛋白能以浓度和时间依赖性的方式转导进入人骨髓间充质干细胞,同时,TAT/LMP-3能成功诱导人骨髓间充质干细胞成骨分化.结论:成功进行了TAT/LMP-3融合蛋白的原核表达、纯化和鉴定,构建的TAT/LMP-3融合蛋白具有入胞转导能力同时保持了骨诱导活性,为其在脊柱融合的进一步应用奠定了实验基础.AIM： To express and purify a recombinant fusion protein TAT/LMP-3 with intraeellular transduction capability and osteogenic activity and observe the cell transduction activity of TAT/LMP-3 and the efficacy of TAT/LMP-3 to induce osteogenic differentiation of human marrow mesenchymal stem cells （hMSCs）. METHODS ： Prokaryotie expression vectors pET43. 1a-TAT/LMP-3 and pET43, 1a-LMP-3 were constructed and used to transform E. coli BL21 （ DE3 ） by gene engineering. The expression of the fusion protein was induced by IPTG. The obtained proteins were purified by Ni-NTA affinity chromatography and identified. Meanwhile, polyclonal antibodies against recombinant LMP-3 protein were prepared. The fusion protein was incubated with hMSCs, and intracellular transduction of the fusion protein was analyzed by Western blotting. In order to analyze the effect of the recombinant protein on osteogenic differentiation of hMSCs, the expression of osteoblast markers was detected. RESULTS： The prokaryotic expression vector carrying pET43.1 a-TAT-LMP-3 fusion gene was constructed successfully, and the expression of TAT/LMP-3 in its soluble form was observed. The purity of the purified TAT/LMP-3 was higher than 90%. Rabbit derived polyclonal antibodies against TAT/LMP-3 were successfully prepared. Western blotting analysis demonstrated that TAT-LMP-3 fusion protein can be transduced into hMSCs in a concentration-and timedependent manner. Meanwhile, TAT/LMP-3 induced osteogenic differentiation of hMSCs successfully. CONCLUSION： Prokaryotic expression, purification and identification of TAT/LMP-3 fusion protein was successful, and TAT/LMP-3 fusion protein exhibited cell transduction capability and osteogenic activity, which provides an experimental basis for utilizing TAT/LMP-3 fusion protein in spinal fusion.

关 键 词：TAT 蛋白转导结构域 重组融合蛋白质类 人骨髓间充质千细胞 诱导成骨 

分 类 号：R392-33[医药卫生—免疫学] R687.34[医药卫生—基础医学]