抗菌肽dermaseptin s4及其突呢变体基因在大肠杆菌中的融合表达.docx

兰州大学硕上学位论文结果发现得到了特异性目的蛋白，透析后经紫外分光光度仪分析，获得了0．2mg／ml 兰州大学硕上学位论文 结果发现得到了特异性目的蛋白，透析后经紫外分光光度仪分析，获得了0．2mg／ml 的融合目的蛋白。 结论：同时成功获得DS4及其突变体K4．S4目的基因片段；成功构建了表达重 组体pGEX-4T-1-DS4)及突变型表达重组体pGEX．4T．1．杨．S4；成功实现了其在大肠 杆菌中的融合表达及纯化，为进一步研究抗菌肽DS4基因的功能和开发新的药物奠 定了基础。 关键词：抗菌肽Dermaseptin S4；重叠延f0PCR；基因克隆；融合表达 Ⅱ 兰州大学硕士学位论文Fusion 兰州大学硕士学位论文 Fusion Expression of The Antimicrobial Peptide Dermaseptin S4 Gene and Its Mutant Gene in Escherichia Oil ABSTRACT OBJECTIVE：To obtain optimum Antimicrobial peptide DS4 gene and its mutant 瞄一S4 gene using gene splicing by overlap extension PCR(SOE PCR)method construct recombinant pGEX-4T．1一DS4 and pGEX-4T-1·K=4一S4，expression and purifi- cation of fusion protein in E．coli BL21． METHODS：Based on amino acid sequence and the preference codon of E．coli in Codon Usage Database，make full use of the DNA Star and Oligo 6．0 bioloigic software to design of DS4 gene and K4一S4 gene，which were amplified through using gene splicing by overlap extension PCR(SOE PCR)method．And they were cloned into vector PUC-18，After restriction analysis and DNA sequencing，the target gene were further ligated with the fusion expression vector pGEX-4T-1，by using restriction analysis and DNA sequencing,the positive recombinant plasmid were transformed to E． coli BL21(DE3)plyS for IPTG induced expression．By optimizing expression condition including different induction time and temperature and IPTG concentration，the results were observed by 15％SDS—PAGE and they revealed that the GST—DS4 and GST．K4．S4 fusion protein were successfully expressed．Harvesting the positive E．coli BL21 and the solubility body were obtained，then purified through GSTrapFF column and obtained the interesting protein 0．2mg／m1． RESULTS： 1．DS4 gene and its mutant K4-S4 gene obtained using gene splicing by overlap extension PCR(SOE PCR)method，which about 103bp(including restriction endonu— cleases site and protective base)． 2．The recombinant PUC．18．DS4 and PUC-18·K4一S4 were constructed successfully, nI 兰州大学硕十学位论文and 兰州大学硕十学