摘要
将大菱鲆(Scophthalmus maxinus)hepcidin基因成熟肽序列经过部分改造后采用PCR方法克隆到毕赤酵母胞内表达载体pGAPZB中,构建了重组胞内表达载体pGAPZB-tbhep6his。用BlnI线性化重组质粒后,电击转化毕赤酵母SMD1168。经过Zeocin筛选和PCR扩增转化子基因组筛选,得到了数株基因工程酵母菌。通过SDS-PAGE和Western blot鉴定,发现目的蛋白与预期蛋白的分子量相吻合,并能与特异抗体antihis特异性结合,表明大菱鲆抗菌肽hepcidin基因在酵母菌中进行了表达。大菱鲆抗菌肽酵母表达载体的构建和表达为进一步研究该抗菌肽的功能并开发新型鱼类饵料奠定了理论基础。
After partly changed by PCR,the hepcidin of turbot (Scophthalmus maxinus)mature peptide sequence was subcloned into pGAPZB vector. Then,a fusion yeast expression vector pGAPZB-tbhep6his was constructed. Subsequently,the vector was digested with BlnI and transformed into SMD1168.After screening on plates with Zeocin,PCR was performed to amplify and identify the genomic DNA of these tranformants.Several recombinat strains that could express the hepcidin gene were found.The expression of fusion protein was detected by SDS-PAGE.Western blotting demonstrated that the recombinant protein could specially react with antihis antibody.These results showed that hepcidin gene was successfully expressed intracellularly in Pichia pastoris.
出处
《长江大学学报（自科版）（中旬）》
CAS
2008年第4期61-66,共6页
Journal of Yangtze University(Nature Science Edition)
基金
国家自然科学基金项目(40376047)
