【摘要】 以含长白猪IFN-γ基因的质粒载体为模板,用原核表达引物扩增了长白猪IFN-γ基因,将其定向克隆至原核表达载体pET28b的EcoRⅠ和XhoⅠ位点,构建了长白猪IFN-γ基因的原核表达载体pET28b-PoIFN-γ。经酶切、PCR鉴定,表明所构建的重组质粒为特异性长白猪IFN-γ基因原核表达质粒。将该重组质粒转化大肠埃希氏菌BL21(DE3)细胞,阳性菌落筛选、SDS-PAGE分析以及Western-blotting分析表明,成功构建了表达重组猪IFN-γ的大肠埃希氏菌基因工程菌株。亚细胞定位分析表明,目的蛋白经原核表达后主要以不溶性包涵体形式存在,其他少量可溶性目的蛋白存在于细胞浆中。更多还原

【Abstract】 The PoIFN-γ gene was inserted into EcoRⅠand XhoⅠmultiple cloning sites of the prokaryotic expression vector pET28b using the expression primers designed according to the sequence of PoIFN-γ gene.The pET28b-PoIFNγ was identified and analyzed by enzymatic digestion and PCR amplification,and was confirmed to be the IFN-γ gene of Changbai porcine.The purified pET28b-PoIFN-γ was transformed into E.coli BL21(DE3) and the recombinant engineering strain was harvested.Identification by positive clone selection,SDS-PAGE and Western-blotting analyses showed that the PoIFN-γ gene was recombinated correctly with pET28b in BL21(DE3) cells.Results confirmed that construction of the recombinant engineering strain of BL21(DE3)/ pET28b-PoIFN-γ was successful.The localization analysis revealed that the rPoIFN-γ was mainly present in the cells in the form of inclusion bodies and a very small portion was soluble in cytoplasm.更多还原