副溶血弧菌ToxR截短体蛋白的表达纯化及其DNA结合活性

Purification and DNA-binding of ToxR truncated protein of Vibrio parahaemolyticus

摘要:

摘要:【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。

Abstract:

Abstract:［Objective］ The DNA-binding domain of ToxR protein of Vibrio parahaemolyticus was expressed using the Escherichia coli BL21λDE3 protein expression system，and its DNA-binding activity was characterized．［Methods］ The fragment of DNA-binding domain at N-terminal of ToxR (ToxR-N) was amplified by PCR from V．parahaemolyticus strain RIMD2210633，and then cloned into the BamHI and Hind III sites of the vector pET28a．The recombinant plasmid pET28a was transformed into BL21λDE3．Over-expression of His-ToxR-N in the LB medium was induced by adding 1 mmol/L IPTG (isopropyl-b-D-thiogalactoside)．The over-expressed protein was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham)，and then the His-tag was removed by using restricted thrombin．The electrophoretic mobility shift assay was used to analyze the DNA-binding activity of ToxR-N to the promoter-proximal DNA regions of calR and VP1687，respectively．The promoter-proximal regions of calR and VP1687 were separately cloned into the pHRP309 vector containing a promoterless lacZ gene．Then，each of the two recombinant LacZ reporter plasmids was transformed into the wide-type strain (WT) and the toxＲ null mutant strain (ΔtoxR)，respectively，to measure the promoter activity (the β-Galactosidase activity) of the target genes in WT and ΔtoxR by using the β-Galactosidase Enzyme Assay System．［Results］The purified ToxR-N protein had the ability to bind to the upstream DNA regions of calR but not VP1687．The LacZ fusion results showed that the transcription of calR and VP1687 was positively and negatively regulated by ToxR in V．parahaemolyticus，respectively． ［Conclusion］The recombinant ToxR-N protein could be used for studying the transcriptional regulation mechanism in V．parahaemolyticus．ToxR fulfills a mechanism of negative regulation of T3SS1 genes by activating the expression of calR through protein- proximal promoter DNA association.