论文部分内容阅读
目的运用基因工程方法获得重组小鼠β-防御素3(MBD3),优化表达条件,并研究其抗菌活性。方法通过反转录PCR方法从小鼠肺组织中扩增Mbd3基因,构建其原核表达质粒,将该质粒转入大肠埃希菌表达菌株Rosetta-gami(2)。优化融合蛋白的表达条件。通过亲和层析、凝血酶酶切、超滤浓缩得到MBD3的重组成熟肽(rMBD3),并采用十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳和Western blot鉴定其正确性。采用微量液体抗菌实验检测rMBD3对多种常见病原性细菌和真菌的抗菌活性。结果获得表达MBD3融合蛋白(fMBD3)的工程菌,fMBD3的表达量可达菌体总蛋白的62.9%,可溶性fMBD3约为1.15g/L。纯化的rMBD3对单细胞真菌和革兰阳性菌显示出较好的抑菌活性。结论 rMBD3具有一定的抗菌活性,为进一步研究该多肽的其他生物学活性及其应用提供参考依据。
OBJECTIVE: To obtain recombinant mouse β-defensin 3 (MBD3) genetically engineered to optimize the expression conditions and to study its antibacterial activity. Methods Mbd3 gene was amplified from mouse lung tissue by reverse transcription polymerase chain reaction (RT-PCR). The prokaryotic expression plasmid was constructed and transfected into E. coli strain Rosetta-gami (2). Optimize fusion protein expression conditions. The recombinant mature MBD3 peptide (rMBD3) was obtained by affinity chromatography, thrombin digestion and ultrafiltration. The recombinant MBM was confirmed by SDS-PAGE electrophoresis and Western blot Sex. The antibacterial activity of rMBD3 against many common pathogenic bacteria and fungi was tested by using trace liquid antibacterial experiment. Results The engineered bacteria expressing MBD3 fusion protein (fMBD3) were obtained. The expression of fMBD3 reached 62.9% of the total bacterial protein and the soluble fMBD3 was about 1.15 g / L. The purified rMBD3 showed good antibacterial activity against single-cell fungi and gram-positive bacteria. Conclusion rMBD3 has some antibacterial activity, which can provide a reference for further study of other biological activity of the polypeptide and its application.