基因重组沙蚕纤溶活性蛋白的克隆、表达和活性检测

 目的从双齿围沙蚕消化道中克隆并表达具有纤维蛋白溶解活性的蛋白。方法提取沙蚕消化道上皮细胞RNA,逆转录获得cDNA。根据丝氨酸蛋白酶家族基因保守序列设计兼并引物,筛选包含丝氨酸蛋白家族保守位点的阳性克隆,进行核苷酸测序。根据该序列利用5′RACE技术进行扩增,将获得的基因片段克隆到pMAL-p2载体中,在大肠杆菌BL21中进行融合表达。表达的融合蛋白经过直链淀粉树脂亲和色谱和DEAE-Sepharose 4B纯化后,纤维蛋白平板法检测其溶栓活性。结果cDNA经5′RACE扩增获得长度为260 bp的基因片段,推测其包含可编码83个氨基酸的完整序列(约9.0×10³);重组pMAL-p2表达出51.0×10³的融合蛋白,经过测定具有明显的溶栓活性。结论从双齿围沙蚕中克隆获得的基因片段表达的融合蛋白具有溶栓活性,有望成为一种新型的纤溶药物。

Cloning and Expression of Recombinant Fibrinolytic Protein from Perinereis aibuhitensis Grube

OBJECTIVE To clone and express a new protease with fibrinolytic activity from Perinereis aibuhitensis Grube digestive tract epithelial cells.METHODS Total RNA of Clamworm digestive tract epithelial cells was extracted by Trizol reagent,and cDNA was obtained,then screened by a specific primer designed according to the amino acid sequence in the conserved domain of serine proteases,the positive clone was sequenced.According to this sequence,the cDNA was amplified using 5′ rapid amplification of cDNA ends,and the product was cloned into pMAL-p2 to construct expression vector,then introduced into Escherichia coli BL21,and expresed by IPTG induction.The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a DEAD-Sepharose 4B column,and its fibrinolytic activity was detected by artificial fibrin plates.RESULTS A DNA band of approximate 260 bp was amplified by RACE,which deduced including a encoding gene of a protetin of 83 amino acids.After being induced by IPTG,the recombinant pMAL-p2 expressed a fusion protein of 51.0×10³,which had obvious fibrinolytic activity detected by artificial fibrin plates methods.CONCLUSION The fusion protein expressed by encoding gene from Perinereis aibuhitensis Grube had obvious fibrinolytic activity,and may be a new thrombolytic agent.