【摘要】 利用基因串联技术表达鸡β-防御素2(AvBD2)重组蛋白,分析其生物结构活性以及蛋白表达量变化。构建了重组串联表达载体p ET-28a-AvBD2op-AvBD2,并将其转化至大肠杆菌BL21中进行诱导表达融合蛋白,经镍柱纯化后进行Western-blot检测、Tricine-SDS-PAGE分析和MALDI-TOF-MS质谱分析。结果显示,单表达工程菌和串联表达工程菌均在9500 u处有特异性条带;采用BCA法测得的单、串联表达工程菌纯化后的蛋白质量浓度分别为0.258和0.769 mg/m L;通过凝胶成像系统分析,单表达工程菌中目标蛋白的表达量约占上清总蛋白的10.3%,而串联表达的约占上清总蛋白的18.3%;MALDI-TOF-MS质谱分析结果表明,单、串联表达蛋白产物的分子质量分别为4 300、7 811 u;琼脂孔穴扩散抑菌法检测证明纯化蛋白对多种微生物具有不同的抗菌活性。更多还原

【Abstract】 The study analyzed the biological structure-activity and the change of protein expression of chicken AvBD2 by gene tandem technology.The constructed recombinant expression vector,p ET-28a-AvBD2op-AvBD2,was transformed into E.coli BL21,which was induced the expression of fusion protein purified by nickel column and analyzed by Western-blot,Tricine-SDS-PAGE electrophoresis and MALDI-TOF-MS mass spectrometry.The specific bands commonly exsisted at the site of 9500 u both in the single and tandem expression engineered bacteria by Western-blot.With the method of BCA,the concentration of protein in single and tandem expressed in the engineered bacteria after purification,was 0.258 mg/m L and 0.769 mg/m L,respectively.By gel imaging systems analysis,the expression of the target protein in single expressed engineering bacteria accounted for 10.3% of the total protein in the supernatant,but the expression of the target protein in tandem expressed engineering bacteria accounted for 18.3% of total protein supernatant.MALDI-TOF-MS mass spectrometry analysis indicated that the molecular masses of single and tandem protein products were 4 300 u and 7 811 u,respectively.AvBD2,the purified protein,was proved to show different antibacterial activities to a variety of microorgnisms by agar diffusion assay.The results indicate that the recombinant tandem chicken AvBD2 increases the expression and molecular weight of the fusion protein, which lays the foundation for the formulation of industrial production.更多还原