【摘要】 目的克隆猪链球菌2型转录调控因子Rex的编码基因进行原核表达和纯化,对其进行生物信息学分析和体外结合活性测定。方法 PCR扩增猪链球菌2型强毒株SS2-1的Rex基因编码区,将其克隆入pET28a质粒中,再将pET28aRex重组质粒转入大肠杆菌表达菌BL21(DE3)中,重组菌株经IPTG诱导后能表达出猪链球菌Rex蛋白;通过体外凝胶迁移实验(EMSA)对Rex蛋白与DNA的结合活性进行分析。结果成功地原核表达并纯化了Rex重组蛋白。Rex蛋白与自身启动子Prex特异性结合,高浓度的NADH抑制两者的结合活性,而NAD~+对结合没有影响。结论通过体外结合试验发现Rex是通过响应NADH/NAD~+平衡来调控自身基因的表达。更多还原

【Abstract】 We cloned and prokaryoticly expressed the gene encoding Redox regulator(Rex)of Streptococcus suis serotype 2and analyzed biological information and in vitro binding activity.The encoding Rexgene of SS2-1strain was amplified by PCR with the designed primers,and then cloned into prokaryotic expression plasmid pET28 a.The recombinant plasmid pET28a-Rex was transformed into E.coli BL21.After induced expression by IPTG,the Rex protein was obtained.The binding activity of Rex protein and DNA was analyzed by gel mobility shift assay(EMSA)in vitro.Purification of recombinant protein Rex was successfully expressed.The presence of NAD+did not have major effect on mobility shift,but addition of NADH almost abolished such a binding activity.By in vitro binding assay,Rex was found to regulate the expression of Prex in response to NADH/NAD+equilibrium.更多还原