小鼠β-防御素30的原核表达和纯化

Prokaryotic Expression and Purification of Recombinant Mouse β-Defensin-30

目的:在原核细胞中表达小鼠β-防御素30(DEFB30),并对表达产物进行鉴定和纯化。方法:用RT-PCR方法扩增小鼠Defb30的cDNA序列,将2个拷贝的cDNA序列串联连入原核表达载体pET28(a),构建重组表达载体pET28(a)-Defb30,并将重组表达载体转化至大肠杆菌Rosetta(DE3),IPTG诱导表达,以Western印迹分析表达产物His-DEFB30,用Ni-NTA亲和柱纯化融合蛋白。结果:构建了Defb30基因的原核表达载体,经IPTG诱导,相对分子质量约15×103的融合蛋白获得表达,Western印迹分析证实此蛋白即为目的蛋白,经Ni-NTA柱亲和纯化,获得了高纯度的融合蛋白His-DEFB30。结论:获得了在大肠杆菌中表达的DEFB30,为研究该蛋白的免疫避孕效果、抗菌活性奠定了基础。

Objective：To prokaryotic express mouse β-defensin-30（DEFB30）.Methods：The Defb30 gene of mouse was cloned by RT-PCR.The two copy of Defb30 cDNA was cloned to pET28（a） vector and transformed in-to E.coli Rosetta（DE3）.The fusion protein was expressed by IPTG induction and characterized by Western blotting analysis.The recombinant protein was purified through Ni-NTA purification system.Results：Prokaryotic expression vector was constructed successfully.SDS-PAGE showed that the molecular weight of the fusion protein His-DEFB30 induced by IPTG was about 15 kD,and the expressed fusion protein was further confirmed by Western blotting analysis.After affinity purification by Ni-NTA,high quality fusion protein was obtained.Conclution：Fu-sion protein DEFB30 was obtained,which provides a basis for further fertility fuctional assay and antibacterial ac-tivity of DEFB30.