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目的构建人成熟转化生长因子β1(hmTGF-β1)的原核表达载体,表达并纯化hmTGF-β1蛋白。方法应用RT-PCR方法获得hmTGF-β1基因,连接至自带6His标签表达载体pET23b,构建原核表达质粒pET23b-hmTGF-β1,诱导表达hmTGF-β1融合蛋白,镍亲合层析法纯化融合蛋白,并对纯化产物进行纯度分析。结果成功构建了hmTGF-β1原核表达质粒pET23b-hmTGF-β1,IPTG诱导蛋白表达,SDS-PAGE电泳分析,在约15kD处出现了一条新的蛋白条带,WesternBlot结果显示该条带能够与His抗体特异性结合。应用Ni-NTA亲和层析法纯化目的蛋白,分析纯度达95%。结论成功构建了hmTGF-β1蛋白的原核表达质粒,并成功表达与纯化hmTGF-β1蛋白,为进一步复性、获得有活性的hmTGF-β1打下基础。
Objective To construct a prokaryotic expression vector of human transforming growth factor β1 (hmTGF-β1) and express and purify the hmTGF-β1 protein. Methods The hmTGF-β1 gene was cloned by RT-PCR and ligated into the native expression vector pET23b. The prokaryotic expression plasmid pET23b-hmTGF-β1 was constructed and the hmTGF-β1 fusion protein was induced. The fusion protein was purified by nickel affinity chromatography. The purity of the purified product was analyzed. Results The prokaryotic expression plasmid pET23b-hmTGF-β1 of hmTGF-β1 was induced by IPTG, and a new protein band appeared at about 15kD by SDS-PAGE. Western Blot showed that this band could bind to His antibody Specific binding. Purification of the target protein by Ni-NTA affinity chromatography, analysis of purity of 95%. Conclusion The prokaryotic expression plasmid of hmTGF-β1 protein was successfully constructed and hmTGF-β1 protein was successfully expressed and purified, which laid the foundation for obtaining active hmTGF-β1 for further refolding.