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人源DCF1基因原核表达载体构建及蛋白分离纯化

DOI: 10.13560/j.cnki.biotech.bull.1985.2014.12.033, PP. 195-200

Keywords: 人源,DCF1,基因,PTD,TAT

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Abstract:

为了深入研究DCF1(DendriticCellFactor1)基因的作用,以质粒pcDNA3.1-DCF1-TAT为模板体外扩增得到片段DCF1-TAT,将测序正确的目的片段克隆入原核表达载体pET32a,转化大肠杆菌Rosetta(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,并优化表达条件。以尿素溶解包涵体蛋白,并优化溶解条件,通过Ni离子亲和层析柱进行纯化,再透析复性目的蛋白。用SDS-PAGE检测目的蛋白的表达和纯化结果,并用Westernblot进行验证。结果表明,重组表达载体pET32a-DCF1-TAT构建成功,并在大肠杆菌Rosetta中成功表达。融合蛋白主要以包涵体形式存在,经过尿素溶解,Ni离子亲和层析柱纯化获得高纯度的目的蛋白。SDS-PAGE和Westernblot检测结果显示蛋白分子量大小与预期结果相符。

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