豹蛙酶的基因克隆及其在大肠杆菌中的表达

根据豹蛙酶的氨基酸序列,采用大肠杆菌偏爱密码子设计引物,重叠延伸PCR法合成出目的基因,约350bp。将得到的片断克隆于载体pGEM-T中并测序,再连接至表达载体pET-22b(+)中,重组质粒转入大肠杆菌Rosetta中。转化的重组大肠杆菌用终浓度1 mmol/L的IPTG诱导外源基因表达,采用Tricine-SDS-PAGE系统,分析目标蛋白主要以包涵体形式存在,其质量分数可达48.8%。利用液相电喷雾串联质谱方式(LC-ESI-MS/MS)鉴定蛋白,通过LCQ DECA XP Plus系统进行蛋白序列的分析,其覆盖率达到35%,确定了其蛋白质一级结构的正确性。

Cloning and expression of recombinant onconase in Escherichia coli

Based on the amino acid sequence of onconase and the preferred codons of the bacterium Escherichia coli, the target gene was synthesized by PCR-driven overlap extension of about 350bp and linked into the pGEM-T vector. After sequencing, the gene was subcloned into the expression vector pET-22b(+) to obtain the recombinant expression vector, which was transformed into Rosetta. Positive colonies were confirmed by PCR and sequencing. In order to check the gene expression in Rosetta, tricine-SDS-PAGE was performed on the target protein. The results showed that the gene was expressed successfully with a content of 48.8%. Moreover, the proposed primary structure of the expressed protein was shown to be correct, with a coverage of 35% , by characterization by LC-ESI-MS/MS with the LCQ DECA XP Plus system.