抗菌肽2IQ3及其突变体的原核表达与纯化  

Prokaryotic expression and purification of antimicrobial peptide 2IQ3 and its mutants

作　　者：马驰[1] 王玉平[1] 谢俊秋[1] 张雪燕[1] 徐攀[1] 杜振亚[1] 倪天治 马如超[1] 韩跃武[1] 

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

出　　处：《兰州大学学报（医学版）》2015年第3期19-22,共4页Journal of Lanzhou University（Medical Sciences）

基　　金：甘肃省卫生行业科研管理项目(GWGL2014)

摘　　要：目的将抗菌肽2IQ3及其突变体基因进行原核表达,经纯化获得目的蛋白。方法根据2IQ3氨基酸序列及大肠杆菌偏爱密码原则设计并合成2IQ3基因编码的核苷酸片段,采用重叠延伸聚合酶链反应法扩增抗菌肽2IQ3及突变体基因,克隆至载体p ET-28a(+)中,构建重组表达质粒,转化至大肠杆菌BL21中,经异丙基硫代半乳糖苷诱导表达,分析重组蛋白的表达形式,纯化重组蛋白。结果构建的抗菌肽2IQ3及其突变体基因所表达的重组蛋白,经十二烷基硫酸钠聚丙烯酰胺凝胶电泳鉴定,有特异性目的条带出现,纯化的重组蛋白纯度达97%左右,浓度为0.502 mg/m L。结论成功构建了抗菌肽2IQ3及其突变体基因的原核表达体系,并在大肠杆菌中进行表达、纯化,为进一步研究其生物学活性奠定了基础。Objective To expression and purify the antibacterial peptide 2IQ3 and its mutant gene in the pro-karyoticcells. Methods Design and synthesize the nucleotide fragment encoded by gene 2IQ3 according to 2IQ3 amino acid sequence and the password policy that E. coli prefers, then amplify antimicrobial peptides 2IQ3 and mutant gene by overlap extension polymerase chain reaction method and clone them into the vector pET-28a （＋） separately, construct the recombinant plasmid, finally transform it into E. coli BL21. After iso-propyl beta-D-thiogalactopyranoside induction, analyze the form of the recombinant protein expression and purify recombinant proteins. Results Recombinant protein expressed by construction of antimicrobial peptides 2IQ3 and its mutant gene is identified by sodium salt-polyacrylamide gel electrophoresis electropho-resis with specific bands, the purity of the purified recombinant protein was about 97%at a concentration of 0.502 mg/mL. Conclusion Successful expression and purification of antimicrobial peptides 2IQ3 and its mutants in E. coli, lay a foundation for further study in its biological activity.

关 键 词：抗菌肽 突变体 大肠杆菌 纯化 

分 类 号：Q78[生物学—分子生物学]