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目的构建血凝素样氧化低密度脂蛋白受体-1(LOX-1)真核表达载体,筛选高表达菌株,为体外获得有活性重组蛋白奠定基础。方法以实验室保存的含有人源LOX-1基因的质粒为模板PCR扩增LOX-1基因,经过TA克隆后亚克隆至酵母表达载体p PICZαA,将获得的重组质粒电转化至毕赤酵母X-33,利用原位双膜法快速检测阳性高表达菌株,最后经过甲醇诱导,采用Western blot检测LOX-1的表达。结果成功构建LOX-1真核表达载体,双膜法筛选到高表达菌株;Western blot结果显示,LOX-1在上清中得到表达。结论 LOX-1基因在毕赤酵母细胞上清中得到表达,为体外获得活性重组蛋白和研究其致动脉粥样硬化功能奠定基础。
Objective To construct the eukaryotic expression vector of hemagglutinin-like oxidized low density lipoprotein receptor-1 (LOX-1) and screen the highly expressed strains for the purpose of obtaining active recombinant protein in vitro. Methods The LOX-1 gene was amplified by PCR using the plasmid containing human LOX-1 gene as a template. After TA cloning, the LOX-1 gene was subcloned into the yeast expression vector p PICZαA. The recombinant plasmid was electroporated into Pichia pastoris X -33, rapid detection of positive expression strains by in situ double membrane method, finally induced by methanol, Western blot detection of LOX-1 expression. Results The LOX-1 eukaryotic expression vector was successfully constructed and screened by double membrane method. The result of Western blot showed that LOX-1 was expressed in the supernatant. Conclusion The LOX-1 gene is expressed in the supernatant of Pichia pastoris, which lays the foundation for obtaining active recombinant protein in vitro and studying its atherogenic function.