人NKp44基因克隆及其在大肠埃希菌中的表达和纯化

Cloning of human NKp44 gene and its expression and purification in E. coli

目的:对人NKp44进行基因克隆及其在大肠埃希菌中重组表达,为进一步研究NK细胞抗肿瘤作用奠定基础。方法:提取人外周静脉血单个核细胞,培养并加入IL-2进行诱导后收集细胞,分离纯化外周血总RNA。用巢式PCR扩增hNKp44片段(约860bp),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-hNKp44重组质粒,分离hNKp44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(+)-hNKp44。转化菌株BL21(DE3)经IPTG诱导,用SDS-PAGE和WesternBlot鉴定表达的重组蛋白。采用His.BindPurificationKit对重组蛋白进行纯化。结果:巢式PCR扩增的DNA片段与hNKp44cDNA大小一致。重组质粒pMD18-T-hNKp44的DNA序列分析显示,克隆的DNA序列与文献报道的hNKp44的cDNA序列一致。SDS-PAGE表明,重组蛋白相对分子质量为35.4×103,其表达量达菌体总蛋白的40%左右。WesternBlot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%的重组蛋白,纯化回收率达40%。结论:已经成功地构建了表达重组hNKp44的工程菌株。

OBJECTIVE： To clone the gene of human NKp44 and express the recombinant human NKp44 in E. coli in order to provide a basis for further study of antitumor immune response of NK cells. METHODS： Peripheral blood mononuclear cells （PBMC） were prepared from a health volunteer＇s peripheral venous blood, cultured and introduced by IL-2, Total RNA was isolated from PBMC. A hNKp44 DNA fragment,with a length of about 860 bp,was amplified from the total RNA by nested PCR and cloned to plasmid pMD18-T,and the cloned DNA fragment was sequenced. The recombinant plasmid pMD18-T-hNKp44 was digested with EcoR Ⅰ and NcoⅠ , then hNKp44 fragment was isolated and inserted to the corresponding restriction site on procaryotic expression vector pET30a（＋）. The recombinant plasmid pET30a（＋）-hNKp44 was identified by restriction analysis 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. The expressed protein was purified by His · Bind Purification Kit. RESULTS： The length of DNA fragment amplified by nested PCR was consistent with that of hNKp44 cDNA. DNA sequencing of pMD18-T-hNKp44 revealed that the cloned DNA sequence was identical to that of reported hNKp44 cDNA. SDS-PAGE proved that expressed product, with a relative molecular weight of 35.4 × 10^3 , contained about 40% of total somatic protein. Western Blot showed that the recombinant protein could specifically bind to anti-His · Tag antibody. The recombinant protein was obtained by purification with 95% of final purity and 40% of recovery rate. CONCLUSION： A recombinant bacterial strain for expressing hNKp44 is successfully constructed.