石莼多糖裂解酶原核表达方法比较

Method Comparison of Prokaryotic Expression of Ulvanlyase

比较不同原核表达载体和受体重组转化表达石莼多糖裂解酶基因的效果。将人工合成的石莼多糖裂解酶基因片段分别酶切接入原核表达载体pColdⅠ质粒和pET-28a(+)质粒,得到重组质粒pColdⅠ-859和pET-28a(+)-859(859代表石莼多糖裂解酶基因),经双酶切测序鉴定后,分别将质粒pColdⅠ-859转入大肠杆菌RP(DE3)感受态细胞,质粒pET-28a(+)-859转入大肠杆菌BL21(DE3)LysS中表达。试验结果显示,前者没有明显表达目的蛋白,后者表达的蛋白经分离纯化、电泳鉴定和Western Blot分析证明目的蛋白表达量较高。本试验经对比研究获得了能充分表达石莼多糖裂解酶的原核表达系统,为批量制备低成本高纯度的石莼多糖裂解酶蛋白提供了参考。

The artificially synthesized ulvanlyase gene was inserted into the prokaryotic expression vectors pColdⅠplasmid and pET-28a(+)plasmid,respectively,and the recombinant plasmid pColdⅠ-859 and pET-28a(+)-859(859 represents ulvanlyase gene)obtained was transferred to Escherichia coli RP(DE3)competent cell,and E.coli BL21(DE3)LysS,respectively,by PCR and sequence analysis to compare the effects of different prokaryotic expression vectors and receptor recombinant transformation on the gene of ulvanlyase after the restriction enzyme identification.The results showed that the former had no obvious target protein,and that the protein expressed by the latter was purified by electrophoresis identification and western blot analysis,showing the high target protein expression.In this experiment,the prokaryotic expression system that fully expressed the ulvanlyase was obtained,which provides a reference for the batch preparation of ulvanlyase protein with low cost and high purity.