人NKp46基因克隆及其在大肠杆菌中的表达和纯化

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·Bind Purification 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。

Cloning of human NKp46 gene and its expression and purification in E.coli

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.then 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.