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目的构建含VLDL-R配体结合结构域融合蛋白表达载体,并在大肠埃希菌中进行表达与纯化。方法利用PCR技术扩增LBR1-8的DNA片段,将其克隆至原核表达载体pET28a(+)中,随后将阳性重组质粒转化到大肠埃希菌Rosetta中,利用IPTG诱导蛋白表达并优化其表达条件,表达产物经Ni+-NTA亲和层析柱纯化回收,并采用SDS-PAGE和Western印迹对目的蛋白进行分析和鉴定。结果构建的重组表达质粒经PCR,酶切和DNA测序鉴定与预期的结果一致,含有重组质粒的表达宿主经过IPTG诱导成功表达了LBR1-8,并经优化确定了最佳的诱导表达条件。结论成功构建pET28a(+)-LBR1-8表达质粒,表达并经纯化得到了LBR1-8。
Objective To construct a fusion protein expression vector containing VLDL-R ligand binding domain and to express and purify it in Escherichia coli. Methods The DNA fragment of LBR1-8 was amplified by PCR and cloned into prokaryotic expression vector pET28a (+). The recombinant plasmid was transformed into Escherichia coli Rosetta. The protein expression was induced by IPTG and its expression conditions were optimized , The expressed product was purified by Ni + -NTA affinity chromatography column, and the target protein was analyzed and identified by SDS-PAGE and Western blotting. Results The recombinant plasmids were identified by PCR, restriction enzyme digestion and DNA sequencing. The recombinant plasmids were successfully expressed in E. coli BL21 (DE3) -induced by IPTG, and the optimal expression conditions were determined. Conclusion The pET28a (+) - LBR1-8 expression plasmid was constructed successfully and expressed and purified by LBR1-8.