雪莲类PEBP基因在大肠杆菌中的表达及鉴定

本实验旨在构建雪莲类PEBP基因原核表达载体,在大肠杆菌中表达并纯化类PEBP基因所编码的蛋白,为进一步研究奠定基础.将雪莲类PEBP基因开放阅读框序列克隆到原核表达载体pET30( )上,转化感受态表达菌株BL21(DE3),低浓度IPTG低温诱导融合蛋白的表达,纯化产物,Western blotting鉴定目的蛋白.IFTG低诱导PEBP,经SDS-PAGE分析,其相对分子量约为28 kD,与预期相符,表达量约占菌体蛋白的26.8%,并且通过亲和层析纯化了重组融合蛋白,Western blotting鉴定为阳性.成功构建了原核表达载体pET-PEBP,获得了高效表达产物,并为进一步研究雪莲类PEBP基因的抗冻功能打下基础.

Expression and Identification of PEBP-like Gene from Saussurea involucrate Kar.er kir in Escherichia coli

This assay was designed to construct the prokaryotic expression vector, investigate the expression of PEBP-like in Escherichia coli and purify its product. The PEBP gene was inserted into the vector pET30a (+). The recombinant vector was transferred into E. coli BL21 (DE3)and induced the expression of protein by low concentration of IPTG and low temperature overnight. After purification, the supernatants were analyzed by SDS-PAGE and the results were identified by Western blotting. After IPTG induction, a new anticipating fusion protein of 28 kD appeared as an expected size, and its product was 26.8% in total protein, the fusion protein was positive by Western blotting. The prokaryotic expression system of PEBP-like is successfully constructed. It lays the foundation for the further application study on the antifreeze characters of the PEBP.