红鳍东方鲀防御素db-1基因的原核表达分析

Cloning and prokaryotic expression of defensin db-1 gene from redfin puffer Takifugu rubripes

采用原核表达方法高效表达红鳍东方鲀Takifugu rubripes防御素db-1基因,并获得该基因的重组表达产物。通过RT-PCR扩增获得红鳍东方鲀防御素db-1基因成熟肽编码序列,将其连入pET32a+载体,构建原核表达载体pET32a-m DB-1,然后将其转入至大肠杆菌表达菌株Rosetta(DE3)感受态细胞中,经IPTG诱导目的基因表达,通过SDS-PAGE和Western Blotting检测该蛋白,并利用Ni-NTA亲和层析纯化该蛋白,最后以Anti-Mus defensin Beta2抗体为探针的Elisa反应鉴定该蛋白。结果表明:红鳍东方鲀db-1基因在大肠杆菌中高效表达,并纯化得到了这种重组防御素融合蛋白。本研究中提供了一种简单、高效获取重组防御素蛋白的方法,可为其应用和生产提供支持。

The cloning and prokaryotic expression of defensin db-1 gene were studied and the recombinant protein of defensin db-1 gene was obtained in redfin puffer Takifugu rubripes by RT-PCR. The mature peptide coding sequences of defensin db-1 gene was inserted into the plasmid pET-32a＋ to construct the prokaryotic expression vector pET32a-m DB-1,which was transformed into Escherichia coli competent cells Rosetta（ DE3） and was expressed after induced by IPTG. The protein expression was detected by SDS-PAGE gel electrophoresis and Western Blotting,and the expressed fusion proteins were purified by affinity chromatography with Ni-NTA. Finally,the fusion proteins were identified by the Elisa with the probe of goat Anti-Mus Defensin Beta2 antibody. The defensin db-1gene was found to be expressed effectively in E. coli,and the recombinant protein has been purified and obtained confidently. The findings provided a simple and efficient way to get the recombinant defensin protein,and a support for the production and application of this protein.