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【目的】利用大肠杆菌BL21λDE3表达系统,表达出有活性的副溶血弧菌(Vibrio parahaemolyticus,VP)ToxR截短体蛋白,为进一步研究ToxR的转录调控机制奠定基础。【方法】以VP基因组DNA为模板,PCR扩增ToxR蛋白DNA结合结构域(ToxR-N)的DNA片段,并将其直接克隆入pET28a中,获得重组质粒;将重组质粒导入大肠杆菌BL21λDE3中,所得菌株经IPTG诱导后能表达出His-ToxR-N蛋白。利用限制级凝血酶切除His-ToxR-N中的His-标签,进而以VP的calR和VP1687为靶基因,通过体外的凝胶阻滞实验(EMSA)验证ToxR-N蛋白的DNA结合活性。分别构建克隆有calR和VP1687上游启动子区的LacZ重组质粒,并将重组质粒转入野生株(WT)和toxR突变株(ΔtoxR)中,通过测定β-半乳糖苷酶活性来比较两株重组菌中靶基因启动子活性,以验证ToxR对calR和VP1687的调控关系。【结果】成功表达出有活性的ToxR-N蛋白,该蛋白对calR启动子区具有结合活性。LacZ结果显示ToxR对calR的转录具有激活作用,而对VP1687的转录具有抑制作用。【结论】所表达的ToxR-N可用于后续的转录调控机制研究;ToxR通过直接激活calR的转录表达,而间接抑制T3SS1相关基因的表达。
【Objective】 The recombinant ToxR truncated protein of Vibrio parahaemolyticus (VP) was expressed in Escherichia coli BL21λDE3 expression system, which laid the foundation for further study on the transcriptional regulation of ToxR. 【Method】 The DNA fragment of ToxR-N was amplified by PCR using VP genomic DNA as a template and directly cloned into pET28a to obtain a recombinant plasmid. The recombinant plasmid was introduced into E. coli BL21λDE3, The resulting strain was induced by IPTG to express His-ToxR-N protein. The DNA-binding activity of ToxR-N protein was verified by in vitro gel retardation assay (EMSA) using restriction-type thrombin to excise His-tag in His-ToxR-N and then target VP of calR and VP1687. The LacZ recombinant plasmids cloned with calR and VP1687 upstream promoter regions were constructed and transformed into wild-type strain (WT) and toxR mutant (ΔtoxR) respectively. The two recombinants were compared by measuring β-galactosidase activity The promoter activity of the target gene in bacteria was examined to verify the regulatory effect of ToxR on calR and VP1687. 【Result】 The active ToxR-N protein was successfully expressed, which has the binding activity to the calR promoter region. LacZ results show that ToxR has an activation effect on the transcription of calR but has an inhibitory effect on the transcription of VP1687. 【Conclusion】 The expression of ToxR-N can be used in the follow-up study of transcriptional regulation. ToxR indirectly inhibits the expression of T3SS1-related genes by directly activating the transcriptional expression of calR.