链霉亲和素-自噬相关基因融合蛋白的原核表达及纯化

Expression and Purification of the Fusion Protein SA-Beclin 1

目的构建链霉亲和素(streptavidin,SA)和自噬相关基因(Beclin 1)重组质粒,并对融合蛋白SA-Beclin 1进行表达和纯化。方法利用基因重组技术将核心SA与Beclin 1序列连接,克隆入pQE80形成pQE80-SA-Beclin 1重组载体,IPTG诱导融合蛋白原核表达,镍亲和凝胶层析柱纯化融合蛋白,Western blot鉴定。结果 PCR成功扩增核心SA活性中心,酶切鉴定和测序均证实重组载体pQE80-SA-Beclin 1构建成功;IPTG诱导后SA-Beclin 1融合蛋白(含His标签)在大肠杆菌中高效表达,SDS-PAGE分析表达的融合蛋白以包涵体为主,经镍亲和凝胶层析柱纯化得到融合蛋白,Western blot鉴定其相对分子量约为72000kD,与预期相符。结论本文成功构建了重组质粒并表达纯化了SA-Beclin 1融合蛋白,为进一步研究SA-Beclin 1的功能及临床应用奠定了基础。

Objective To construct recombinant plasmid of streptavidin （SA） and autophagy-related genes Beclin1 fusion gene （Beclin 1）and to express and purify SA-Beclin1 fusion protein.Methods The natural core SA sequence and beclin1 gene were cloned into pQE80 to form the recombinant plasmid pQE80-SA-Beclin 1.The expression of fusion protein was induced by IPTG,purified by Ni-NTA agarose and detected by Westernblot.Results The natural core SA was amplified by PCR,restriction enzyme digestion and sequencing results showed that the recombinant plasmid pQE80-SA-Beclin1 was successfully constructed.The fusion protein induced by IPTG was efficiently expressed in E.coli.Fusion protein was detected by SDS-PAGE and purified by Ni-NTA agarose with a molecular weight of 72 000 kD identified by Western blot.Conclusion The SA-Beclin1 fusion protein was successfully expressed and purified,which lay a good basis for further functional research and clinical application of beclin1.