人源弹性蛋白酶基因真核载体构建及表达

Construction and expression of eukaryotic vector of elastase gene in human

从人体肾脏组织中提取的总RNA中通过反转录PCR(RT-PCR)扩增得到了ELA2B基因,并将其克隆到pGEM-Teasy载体中。然后用限制性内切酶EcoRⅠ和NotI对重组质粒pGEM-Teasy/ELA2B和毕赤酵母质粒pPIC9K进行双酶切,并且通过PCR技术去除ELA2B的信号肽,成功构建了真核表达载体pPIC9K/ELA2B,并将其转入毕赤酵母表达宿主GS115中。得到150株转化子,经含不同浓度G418的YPD平板筛选获得20株高拷贝转化子。甲醇诱导表达后,发酵液上清经SDS-PAGE检测,获得了大小约为28ku的目的条带。通过酪蛋白平板检测发酵液上清,出现透明圈,初步证明获得了有生物活性的弹性蛋白酶。

The human ELA2B gene was cloned from total RNA which was extracted from kidney of human by reverse transcription polymerase chain reaction （RT-PCR）,then a recombinant plasmid pGEM-T easy/ELA2B was obtained. After the recombinant plasmid pGEM-T easy/ELA2B and pichia expression plasmid pPIC9K digesting with EcoR I and Not I,and removing the signal peptide of ELA2B by PCR,the recombinant eukaryotic expression plasmid pPIC9K/ELA2B were successfully constructed and transformed into pichia host GS115. 101 transformants were selected on the MD agar plates,and 16 highly expression transformants were screened by YPD plates containing G418. After expression of recombinant elastase in shaking flask by methanol induction,it was found the size of recombinant elastase was approximately 28ku by SDS-PAGE analysis. The casein protein could be hydrolyzed by fermentation supernatant which initially indicated that the expression product had biological activity.