人源弹性蛋白酶基因原核载体构建及表达

Construction and Expression of Prokaryotic 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-T easy载体中。然后用限制性内切酶BamH I和HindⅢ对重组质粒pGEM-T easy/ELA2B和大肠杆菌E.coli表达质粒pET30a进行双酶切,成功构建了原核表达载体pET30a/ELA2B。将重组质粒转化大肠杆菌表达宿主BL21中,通过PCR和表型鉴定表明,ELA2B基因已经整合到大肠杆菌BL21染色体上。用IPTG对重组的大肠杆菌BL21进行诱导表达,得到的蛋白经SDS-PAGE分析结果表明:菌体中含有一明显特异性蛋白,大小为28 kD。经包涵体复性后在弹性蛋白酶活性检测平板上发现有代表弹性蛋白酶作用的透明圈产生,可初步证实表达产物具有生物活性。

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 E.coli expression plasmid pET30a digesting with BamH I and Hind Ⅲ,the recombinant prokaryotic expression plasmid pET30a/ELA2B were successfully constructed and transformed into E.coli host BL21.PCR and phenotype analysis showed that ELA2B has been integrated into E.coli host BL21 genome.After expression of recombinant elastase in shaking flask by IPTG induction,we found the size of recombinant elastase was approximately 28kD by SDS-PAGE analysis.After refolding of inclusion body,transparent circle was found in activity monitor flat panel,which initially suggested that the expression product has biological activity.