VLDL-R配体结合结构域的克隆与表达

Cloning and Expression of Very Low Density Lipoprotein Receptor's Ligand Binding Domain

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 the expression vector of VLDL-R＇s ligand binding domain fusion protein and study its expression and purification in E. coli. Methods The DNA fragment of LBR1-8 was amplified from VLDL-R cDNA recombinant plasmid by PCR and was inserted into plasmid pET28a （ ＋ ） to construct the prokaryotic expression plasmid pET28a （ ＋ ） -LBR1-8. The positive recombinant plasmid was transformed into E. coli strain Rosetta and then was induced by IPTG, with the expression conditions optimized. The fusion protein was purified by Ni^＋ -NTA chromatography column, analyzed and identified by SDS-PAGE and Western blotting. Result The recombinant expression plasmid was identified by PCR, digested with restricted endoenzymes and subjected to DNA sequencing. It was found to be consistent with the predicted result. The host cells containing the recombinant plasmid that successfully expressed LBR1-8 after being induced by IPTG and the optimal inducing conditions were determined. Conclusion The prokaryotic expression and plasmid pET28a （ ＋ ） LBR1-8 was successfully constructed and the target recombinant protein LBR1-8 was expressed and purified, which lays a foundation for the study on the functions of VLDL- R function and its application, especially on its ligand recognition and binding characteristics.