皮蝇素A基因的克隆及其毕赤酵母表达载体的构建

Cloning of Hypodermin A Gene of Hypoderma Species and Construction of Pichia pastoris Expression Vector

提取采自甘肃玛曲皮蝇一期幼虫的总RNA,合成cDNA,根据设计的特异引物,利用PCR技术扩增出皮蝇素A(Hypodermin A,HA)基因片段,用XhoⅠ、XbaⅠ消化,将消化后的目的基因HA纯化并回收,与经同样的酶消化后并纯化回收的载体pPICZa连接并转化,获得重组表达质粒pPICZa-HA,经PCR、酶切、测序表明,目的基因插入位置、方向和阅读框正确。

The total RNA of first-stage larvae from Hypoderma species colleted from Maqu Gansu was isolated and used for synthesis of cDNA. According to specific primer, HA gene was amplified with PCR and digested with Xho Ⅰ and Xba Ⅰ . This gene was ligated into vector pPICZa digested with Xho Ⅰ and Xba Ⅰ , and got recombinant plasmid pPICZa-HA. The recombinant plasmid was identified by restriction anailysis and PCR and DNA sequencing. Positive recombinants were selected, which could be further expressed in Pichia pastoris cell.