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。
The PoIFN-λ, gene was inserted into EcoR I and Xho I multiple cloning sites of the prokaryotic expression vector pET28b using the expression primers designed according to the sequence of PoIFN-λ, gene. The pET28b-PoIFN-λ, was identified and analyzed by enzymatic digestion and PCR amplification, and was confirmed to be the IFN-λ, gene of Changbai porcine. The purified pET28b-PoIFN-λ, 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-λ, gene was recombinated correctly with pET28b in BL21 （DE3） cells. Results confirmed that construction of the recombinant engineering strain of BL21 （DE3）/ pET28b-PoIFN-λ, was successful. The localization analysis revealed that the rPoIFN-λ, was mainly present in the cells in the form of inclusion bodies and a very small portion was soluble in cytoplasm.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2006年第1期46-51,共6页
Chinese Veterinary Science
