大肠杆菌中融合表达人β防御素-3基因的研究(英文)

Research of Fusion Expression of Human β-Defensin-3 in Escherichia coli

目的为了在大肠杆菌中融合表达人β防御素-3基因。方法根据大肠杆菌对精氨酸密码子使用的偏爱性,设计搭桥引物,并通过PCR扩增法合成了人β防御素的全基因序列,克隆进pGEX-4T-2中构建pGEX-4T-2-hBD-3融合表达载体。将表达载体转化E.coli宿主菌DH5α,进行IPTG诱导表达。将菌体反复冻溶使细胞膜穿孔,释放可溶性蛋白。融合蛋白GST-hBD-3经凝血酶切割。结果研究得到了重组人防御素蛋白,琼脂孔穴扩散抑菌法检测表明,重组人β防御素3对金黄色葡萄球菌有抗菌活性,抑菌效价为0.843 U。结论人β防御素-3基因在大肠杆菌中得到了融合表达。

Objective To achieve the fusion expression of the entire human beta-defensin-3 （hBD-3） gene. Method We synthesized two oligonucleotide primers according to the eodon preference of Escherichia coll. The gene was cloned into pGEX- 4T-2 to establish the pGEX-4T-2-hBD-3 as the fusion expression vector by PCR. Transformed into E. coli strain DH5a, the express vector was induced and expressed by IPTG. The fusion protein GST-hBD-3 was obtained by repeated cycles of freezing and thawing, cut by thrombin to attain the recombinant hBD-3 protein. Result The result of the antibacterial peptide agarose diffusion assay shows the antibacterial activity of the rhBD-3 against the S. aureus exists, and it reaches 0.843U. Conclusion The fusion expression of the hBD-3 gene is successful.