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目的设计、合成天蚕素AD(CAD)基因序列,与内含肽(Intein)融合表达后通过重组蛋白自剪切作用获得C端酰胺化的CAD,并与抗生素配伍使用。方法根据大肠埃希菌密码子偏好性对CAD基因序列进行改造,人工合成目的基因,构建重组质粒pCold-SUMO-CAD-Intein,转化大肠埃希菌,诱导表达重组蛋白,经镍柱亲和层析纯化,后用DTT及肠激酶切割,利用分子筛纯化获得酰胺化CAD,并进行电泳及质谱鉴定。采用微量肉汤法检测重组CAD抑菌活性及其与不同抗生素按不同比例配伍使用的抑菌活性。结果 PCR扩增目的基因片段为132bp,与预期一致。质谱显示表达的CAD分子质量单位为3.949ku,与理论值相符。所得CAD抗菌肽对常见革兰阴性菌抗菌力强,配伍抗生素使用能发挥协同抑菌作用。结论成功构建CAD基因重组质粒pCold-SUMO-CAD-Intein,通过与内含肽(Intein)融合表达,经分子内的自剪切作用直接获得C末端酰胺化的抗菌肽,与抗生素配伍使用,能显著降低抗生素用量,具有作为抗生素替代物的应用潜力。
Objective To design and synthesize the AD (CAD) gene sequence of cecropin and synthesize C-terminal amidated CAD by self-shearing of the recombinant protein after fusion with intein, and to use it in combination with antibiotics. Methods According to the codon preference of Escherichia coli, the CAD gene sequence was modified. The recombinant plasmid pCold-SUMO-CAD-Intein was constructed by artificial synthesis. The recombinant plasmid was transformed into Escherichia coli. After purification, digested with DTT and enterokinase, the molecular sieve was used to obtain amidated CAD, and electrophoresis and mass spectrometry identification. Antibacterial activity of recombinant CAD was tested by micro broth method and its antibacterial activity was tested with different antibiotics in different proportions. Results The target gene fragment amplified by PCR was 132bp, which was consistent with the expectation. Mass spectrometry showed the expressed CAD molecular mass unit was 3.949ku, consistent with the theoretical value. The resulting CAD antimicrobial peptides on the common gram-negative bacteria antibacterial strong compatibility of antibiotics can play a synergistic antimicrobial effect. CONCLUSION: The recombinant plasmid pCold-SUMO-CAD-Intein of CAD gene was constructed successfully and the C-terminal amidated antimicrobial peptide was directly obtained through the intramolecular self-shearing by the fusion with intein (Intein) Significantly reduce the amount of antibiotics, with potential as a substitute for antibiotics.