抗菌肽Dermaseptin S4及其突变体基因在大肠杆菌中的表达  

Expression of Antimicrobial Peptide Dermaseptin S4 Gene and Its Mutant in E. coli

作　　者：张庆华[1] 韩跃武[1] 谢俊秋[1] 卢天龙[1] 任惠子[1] 冯惟萍[1] 

机构地区：[1]兰州大学基础医学院生物化学与分子生物学研究所,兰州730000

出　　处：《中国生物制品学杂志》2008年第4期269-272,共4页Chinese Journal of Biologicals

基　　金：国家高技术研究发展计划(863计划)项目(2006AA10A208-1-4)

摘　　要：目的构建表达抗菌肽Dermaseptin S4(DS4)及其突变体K4-S4的工程菌,并表达目的蛋白。方法采用重叠延伸PCR法设计和扩增抗菌肽DS4及K4-S4基因,定向克隆入载体pUC-18中,经酶切、测序鉴定,重组至表达载体pGEX-4T-1中,构建抗菌肽DS4及K4-S4基因表达载体,转化大肠杆菌BL21(DE3)plyS,经IPTG诱导表达,12%SDS-PAGE鉴定。结果构建的抗菌肽DS4及K4-S4基因工程菌所表达的目的蛋白,经SDS-PAGE鉴定可见相对分子质量34 000的特异性目的条带,37℃诱导5 h的蛋白表达量最高,且多数以包涵体形式存在。结论已成功构建抗菌肽DS4及K4-S4工程菌,并表达目的蛋白,为进一步研究其功能和应用奠定了基础。Objective To construct a recombinant E. coli strain for expression of antimicrobial peptide Dermaseptin S4 （DS4） gene and its mutant K4-S4 and express the target proteins. Methods Design and amplify DS4 and K4-S4 genes by overlap extension PCR and clone into vector pUC-18 respectively. Identify the constructed recombinant plasmids pUC18-DS4 and pUC18-K4-S4 by restriction analysis and sequencing, and insert the obtained target genes into expression vector pGEX-4T-lrespectively. Transform the constructed recombinant plasmids pGEX-4T-1-DS4 and pGEX-4T-1-K4-S4 to E. coli BI21 （DE3） plyS for expression under induction of IPTG. Identify the expressed product by 12%SDS-PAGE.Results DS4 and K4-S4 proteins, both with relative molecular masses of 34 000, were expressed. The expressed product mainly existed in a form of inclusion body. The expression level reached a peak value 5 h after induction. Conclusion A recombinant E. coli strain for expression of D4 and K4-S4 genes was successfully constructed,and the target protein was expressed, which laid a foundation of further study on function and application of D4 and K4-S4 proteins.

关 键 词：抗菌肽 DERMASEPTIN S4 突变体 大肠杆菌 表达 

分 类 号：Q78[生物学—分子生物学]