重组人β防御素3在大肠杆菌中的表达和活性分析

防御素是生物界广泛分布的一类低分子短肽,具有广谱高效的杀菌、抗肿瘤作用,并且不易使微生物产生抗药性,具有很高的应用价值,其中最引人注目的是β防御素1,2].人β防御素3(humanβ-defensin3,hBD3)是最近发现的第3种人源性β防御素,与其它人防御素相比,在抗菌活性等方面具有明显优势,是所有防御素中抗菌能力最强的之一3~7],具有独特的研究和开发价值.为了得到高效表达hBD3的工程菌株,本实验按照细菌对密码子的偏爱,人工合成了hBD3的寡核苷酸片段,构建了其表达载体.经IPTG诱导、分离纯化和肠激酶切割,得到了与天然hBD3活性基本相同的…

Expression of Recombinant Human β-Defensin 3 in E.coli and Its Antimicrobial Activity Analysis

Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,four pairs of oligonucleotide with cosmic site were synthesised using E.coli biased codons according to the amino acid sequence of β-defensin 3, connected and amplified by PCR. The PCR product encoding human β-defensin 3 was cloned into pET30a vector.The recombinant vector was transformed into E.coli BL21(DE3)PlysS and the expression was induced by IPTG. The recombinant fusion protein was analyzed by SDS-PAGE and purified by affinity column. The mass of the fusion protein consisted of 30.9% in total bacteria proteins. The recombinant fusion protein was digested by enterokinase, resulting in the recombinant hBD-3. Antimicrobial activity analysis showed that both recombinant hBD-3 fusion protein and recombinant hBD-3 had similar potency as the native protein in suppressing growth of both gram positive bacteria S.aureus and gram negative one E.coli in a dose dependent manner.