【摘要】 目的:对人NKp46进行基因克隆并探讨其在大肠杆菌中重组表达和纯化。方法:用RT-PCR法自人外周血单个核细胞总RNA扩增hNKp46片段(约900 bp),克隆至质粒载体pMD18-T,并对克隆的DNA片段进行序列分析。用限制酶EcoRⅠ和NcoⅠ消化pMD18-T-hNKp46重组质粒,分离hNKp46片段,并插入原核表达载体pET30a(+)的相应限制酶位点,酶谱分析鉴定重组表达载体pET30a(+)-hNKp46。转化菌株BL21(DE3)经IPTG诱导,用SDS-PAGE和Western Blotting鉴定表达的重组蛋白。采用His.BindPurification Kit对重组蛋白进行纯化。结果:RT-PCR扩增的DNA片段与hNKp46 cDNA大小一致。重组质粒pMD18-T-hNKp46的DNA序列分析显示,克隆的DNA序列与文献报道的hNKp46的cDNA序列一致。SDS-PAGE表明,重组蛋白相对分子质量为38.5 kD,其表达量达菌体总蛋白的40%左右。Western Blotting分析显示,重组蛋白能特异地与抗His.Tag抗体结合。纯化得到纯度为95.5%的重组蛋白,纯化回收率达40%。结论:成功构建了人NKp46表达载体,并获得了稳定表达的工程菌株,纯化了重组蛋白。更多还原

【Abstract】 Objective:To clone the gene of human NKp46,express and purify the recombinant human NKp46 in E.coli.Methods:A hNKp46 DNA fragment,with a length of about 900?bp,was amplified from the total RNA of peripheral blood mononuclear cells by RT-PCR and cloned to plasmid pMD18-T,and then the cloned DNA fragment was sequenced.The recombinant plasmid pMD18-T-hNKp46 was digested with EcoRⅠ and NcoⅠ,andthen hNKp46 fragment was isolated and inserted to the corresponding restriction site on procaryotic expression vector pET30a(+).The recombinant plasmid pET30a(+)-hNKp46 was identified by enzymogram and transformed to(E.coli) BL21(DE3),and then its expression was induced by IPTG.The expressed product was identified by SDS-PAGE and Western Blotting,and the expressed protein was purified by His·Bind Purification Kit.Results:The length of DNA fragment amplified by RT-PCR was consistent with that of hNKp46 cDNA.DNA sequencing of pMD18-T-hNKp46 revealed that the cloned DNA sequence was identical to that of reported hNKp46 cDNA.SDS-PAGE proved that expressed product,with a relative molecular weight of 38.5?kD,contained about 40% of total somatic protein.Western Blotting showed that the recombinant protein could specifically bind to anti-His·Tag antibody.The recombinant protein was obtained by purification with 95.5% final purity and 40% recovery rate.Conclusion:A recombinant bacterial strain for expressing hNKp46 is successfully constructed,and its recombinant protein is purified.更多还原