人LOX-1基因毕赤酵母表达载体的构建与表达研究

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摘要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pPICZαA，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。

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	刘京
	于田田
	陈玉华
	李敬达
	陶娅妮
	李明英
	王晓
	陈勇
	迟彦

关键词 ：血凝素样氧化低密度脂蛋白受体-1, 真核表达载体构建, 毕赤酵母, 动脉粥样硬化

Abstract：Objective To construct LOX-1 eukaryotic expression vector and screen high expression strain,lay the foundation for obtaining active recombinant LOX-1 protein in vitro.M ethods LOX-1 gene was amplified by PCR with the plasmid containing human LOX-1 gene as a template.After TA clone,LOX-1 gene was subcloned into eukaryotic expression vector pPICZαA to obtain pPICZαA-LOX-1 recombinant plasmid.After double digestion identification,the positive recombinant plasmid was electro transformed into Pichia pastoris X-33.Situ double membrane hybridization method was used to rapidly screen positive highly expressed strains.Finally,Western blotwas used to test its expression. Results LOX-1 recombinant expressed vector was constructed and highly expressed strain was screened out successfully.Western blot results showed that the LOX-1 recombinant protein was expressed in the cell culture supernatant after induction by methanol.Conclusion LOX-1 gene is expressed in Pichia pastoris cell supernatant.This study laies a foundation for obtaining active recombinant LOX-1 protein in vitro and studying its role in atherosclerosis.

Key words： LOX-1 Vector construction Pichia pastoris Atherosclerosis

基金资助:国家自然科学基金资助项目（81202536）；
辽宁省大学生创新训练项目（201511258040）

通讯作者: 迟彦（1974-），女，辽宁大连人，博士，副教授，研究方向：糖脂代谢

作者简介: 刘京（1994-），女，辽宁锦州人，大连大学生命科学与技术学院2013级生物工程专业本科在读

引用本文:

刘京;于田田;陈玉华;李敬达;陶娅妮;李明英;王晓;陈勇;迟彦. 人LOX-1基因毕赤酵母表达载体的构建与表达研究[J]. 中国当代医药, 2016, 23(32): 4-6转10.
LIU Jing;YU Tian-tian;CHEN Yu-hua;LI Jing-da;TAO Ya-ni;LI Ming-ying;WANG Xiao;CHEN Yong;CHI Yan. Study of the establishment of human LOX-1 gene Pichia expression vector and its expression. 中国当代医药, 2016, 23(32): 4-6转10.

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http://dangdaiyiyao.com/CN/ 或 http://dangdaiyiyao.com/CN/Y2016/V23/I32/4

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