重组人酸性成纤维细胞生长因子-穿膜肽融合蛋白的克隆表达、纯化及活性检测

Cloning,expression,purification and bioactivity assay of fusion protein of rhaFGF-tat

目的:高效表达并纯化具有生物学活性的重组人酸性成纤维细胞生长因子(aFGF)-穿膜肽(tat)融合蛋白,为进一步开发基因工程药物提供实验依据。方法:通过双酶切方法,从质粒pMD18-T-aFGF-tat中获得aFGF-tat基因片段,并将aFGF-tat融合基因与原核表达载体pET3c连接后转化到大肠杆菌BL21(DE3)宿主细胞中,通过IPTG诱导获得可溶性表达,将融合蛋白经CM离子交换层析与肝素亲和层析纯化,MTT法检测aFGF-tat融合蛋白对NIH3T3细胞的促增殖作用。结果:经酶切和基因测序证实获得的重组人aFGF-tat基因序列与GenBank报道的完全一致,构建的pET3c-aFGF-tat表达载体在大肠杆菌BL21(DE3)中成功表达aFGF-tat融合蛋白,蛋白表达量在15%以上,经SDS-PAGE证实其相对分子质量与理论预期值相符,经CM离子交换层析与肝素亲和层析纯化后蛋白纯度高于95%,Western blotting分析表达产物与人aFGF多克隆抗体具有特异结合能力,并能够促进NIH3T3细胞增殖。结论:成功表达重组人aFGF-tat融合蛋白工程菌,获得aFGF-tat纯蛋白,并证实aFGF-tat融合蛋白具有促细胞增殖活性。

Objective To express and purify the fusion protein of rhaFGF-tat with biological activity,and provide a foundation for development of an genetically engineered drug.Methods The aFGF-tat gene fragment through digesting the pMD18-T-aFGF-tat was obtained,then the recombinant plasmid was transformed into E.coil BL21 （DE3）and induced by IPTG.The protein was purified by CM-Sepharose FF cation exchange and heparin-Sepharose FF affinity.The activity of aFGF-tat was detected with NIH 3T3proliferation assay.Results Acquired gene fragments of aFGF-tat identified by digestion and DNA sequencing were coincident with the human aFGF-tat reported in GenBank.The recombinant vector of pET3c-aFGF-tat was constructed successfully.SDS-PAGE result proved that aFGF-tat fusion protein with a relative molecular mass of about 19 102was expressed.The purity of fusion protein was more than 95%,Western blotting results showed that the expressed products had specific reaction with anti-human aFGF polyclonal antibody and was able to promote the proliferation of NIH 3T3cells. Conclusion aFGF-tat fusion gene is expressed in E.coil and purified successfully,and the aFGF-tat fusion protein can promote the proliferation of cells.