小鼠β-防御素3的原核表达、表达条件优化及其抗菌活性

Prokaryotic expression,condition optimization and antifungal activities of mouse β-defensin-3

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 the recombinant mouse β-defensin-3（MBD3） peptide by genetic engineering,optimize the expression condition and detect the antifungal activity of MBD3. MethodsThe full-length of Mbd3 gene was amplified from the lung tissues of BALB/c mouse after injection of lipopolysaccharide by reverse transcription-PCR to construct the prokaryotic expression plasmid,which was cloned into the pET-32a（＋） expression vector and then transferred into Escherichia coli expression strain Rosetta-gami（2）.The expression conditions of fusion protein were optimized.The recombinant mature peptide of MBD3（rMBD3） was obtained by affinity chromatography,thrombin digestion and ultrafiltration,and identified by SDS-PAGE electrophoresis and Western blot.The antimicrobial and antifungal activities of rMBD3 were detected by micro-fluid test. Results The Rosetta-gami（2）-pET32a（＋）/MBD3 was successfully constructed.Under the optimal conditions,the fMBD3 expression occupied 62.9% of the total bacterial protein,and soluble fMBD3 was about 1.15g/L.The rMBD3 was correctly expressed by SDS-PAGE and Western blot.The inhibitory activity of purified rMBD3 against monoplast eumycete and Gram-positive bacteria was confirmed. Conclusion The rMBD3 has inhibitory activity against some pathogens,which may provide an useful foundation for further study of its other biological activity.