旋毛虫醛缩酶基因的克隆表达及重组蛋白酶比活性的分析

Cloning and expression of aldolase gene fromTrichinella spiralis and analysis of specific activities of the recombinant protease

根据旋毛虫醛缩酶(aldolase,ALD)基因序列设计1对特异性引物,以肌幼虫总RNA为模板,通过RT-PCR扩增肌幼虫的ALD基因。将该基因克隆到pMD18-T载体后进行序列测定和分析,再将该基因亚克隆到pET-32a(+)载体中,测序验证后转化大肠杆菌BL21(DE3),IPTG诱导后用SDS-PAGE进行分析。结果显示,该基因与GenBank中旋毛虫醛缩酶基因的相似性高达99%。该基因在大肠杆菌中获得了表达,表达的融合蛋白大小约为57ku,重组蛋白在菌体上清和沉淀中均有存在。Western-blot分析显示,重组蛋白可被人工感染旋毛虫的大鼠血清所识别。通过3-磷酸甘油醛脱氢酶偶联法测定该重组酶的比活性,发现该重组蛋白的最佳反应温度和pH值分别为35℃和7.0。结果表明,该重组蛋白具有一定的抗原性和酶比活性。

Based on the aldolase(ALD)gene sequence(XM_003374234)of Trichinella spiralis,apair of primers was designed.The gene,1.1kb,was amplified by RT-PCR and cloned into pMD18-T vector. After sequencing,the gene was sub-cloned into the prokaryotic expression vector pET-32a(+)and transformed into Escherichia coli BL21(DE3)followed by induction with IPTG.In result,this gene was 99% similarity with that of T.spiralis ALD gene available in GenBank.SDS-PAGE analysis indicated that the recombinant protein was expressed with the molecular weight of 57ku.The result of Western-blot showed that the recombinant protein could be recognized by serum from the rat infected with T.spiralis,which showed that the expressed protein had reactionogenicity.In the biochemical activity assay,it was found that the purified recombinant protein exhibited specific enzymatic activities,and the optimum reaction temperature and pH for the recombinant enzyme was 35℃and 7.0,respectively.