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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 express prokaryotic peptide 2IQ3 and its mutants in prokaryotic cells and purify the target protein. Methods The nucleotide sequence of 2IQ3 gene was designed and synthesized according to the amino acid sequence of 2IQ3 and the preferred codon of Escherichia coli. The antimicrobial peptide 2IQ3 and its mutant gene were amplified by overlap extension polymerase chain reaction (PCR) and cloned into vector pET-28a (+ ), The recombinant expression plasmid was constructed, transformed into E. coli BL21, induced by isopropylthiogalactoside, analyzed the expression of the recombinant protein and purified the recombinant protein. Results The recombinant protein expressed by the antimicrobial peptide 2IQ3 and its mutant gene was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A band with specific purpose appeared. The purity of the purified recombinant protein was about 97% The concentration of 0.502 mg / m L. Conclusion The prokaryotic expression system of the antimicrobial peptide 2IQ3 and its mutant gene was successfully constructed and expressed in Escherichia coli for purification, which laid the foundation for further study of its biological activity.