辣根过氧化物酶基因真核表达载体的构建

Construction of prokaryotic expression vector of horseradish peroxidase gene

从辣根侧根中提取总RNA,以OligodT(18)为引物,通过反转录获得了辣根总mRNA的cDNA。以获得的cDNA为模板,利用设计的一对特异寡核苷酸引物P1和P2进行PCR扩增,得到了辣根过氧化物酶(Horseradish Peroxidase,HRP,EC1.11.1.7)同功酶C2的结构基因(hrpC2)。将hrpC2与真核表达质粒载体pPICZα-A分别用限制性内切酶EcoRI和Xbal双酶切后连接,构建了重组质粒表达载体pPICZα-A-hrpC2。将pPICZα-A-hrpC2转化大肠杆菌筛选阳性克隆并测序。测序结果显示,pPICZα-A-hrpC2重组质粒构建成功。利用BlastN程序进行DNA序列分析,显示该序列为目的ORF框,无移码突变;与Fujiyama.K,Takemura.H等报道的hrpC2序列[D90115.1]有95%的同源性。

The Horseradish Peroxidase C2 gene （ hrpC2 ） was cloned from Armoracia rusticana （Horseradish） by means of reverse transcription.And the hrp was cloned into pPICZα-A vector successfully, after the hrpC2 and pPICZα-A plasmid vector was digested separately using EcoRI and XbaLThen,the constructed pPICZα-A-hrpC2 plasmid vector was transformed into E.coli TOP10, and the positive recombinant clones were selected and checked.The result of sequence analyzing also indicted that the pPICZα-A-hrpC2 was obtained successfully,and the hrpC2 sequence from Armoracia rusticana was homologous by 95% to hrpC2 sequence [ D90115.1] reported by Fujiyama. K, Takemura.H.