肠炎沙门菌非编码小RNA RyhB调控SopE蛋白的表达研究

为研究肠炎沙门菌(SE)50336非编码小RNA(RyhB-1、RyhB-2)对毒力蛋白SopE的调控作用,利用pET原核表达系统表达SE50336 SopE蛋白,免疫BALB/c小鼠获得SopE蛋白的特异性多克隆抗体(多抗)并分析制备血清的特异性。通过Western-blot检测野生株SE50336、RyhB单缺失株(SE50336 ΔryhB-1、SE50336 ΔryhB-2),以及RyhB双缺失株(SE50336 ΔryhB-1ΔryhB-2)中SopE蛋白的表达水平,分析RyhB对SopE的调控作用。结果显示:成功构建重组质粒pET28a-sopE,转化BL21(DE3)感受态细胞后,在IPTG 0.3 mmol/L、温度37℃、转速220 r/min、诱导时间4 h的条件下,SopE蛋白在包涵体中表达;Western-blot结果显示制备的多抗可特异性检测肠炎沙门菌SopE蛋白;与野生株相比,RyhB单缺失株SopE蛋白表达量下降,双缺失株下降更明显,表明RyhB-1和RyhB-2均可上调SopE蛋白的表达。以上结果为进一步研究RyhB-1和RyhB-2对SopE的调控作用机制奠定基础。

Non-coding Small RNA RyhB Regulates the Expression of SopE Protein in Salmonella Enteritidis

To study the regulation of the non-coding small RNA RyhB-1 and RyhB-2 to virulence-related protein SopE in Salmonella enterica serovar Enteritidis(SE)50336,SopE was expressed using pET prokaryotic expression system,and the polyclonal antibodies(PcAbs)against SopE were prepared by immunizing BALB/c mice.The specificity of PcAbs was then analyzed.The expressions of SopE in wild-type SE50336,RyhB deletion mutants(SE50336 ΔryhB-1,SE50336 ΔryhB-2),and(SE50336 ΔryhB-1ΔryhB-2)were used for Western-blot to analyze the regulation of RyhBs to SopE.The results showed that recombinant plasmid pET28 a-sopE was constructed and transformed into BL21(DE3)competent cells successfully.The expression of fusion protein SopE was induced by 0.3 mmol/L IPTG at 37℃ for 4 h with rotation of 220 r/min.The recombinant protein SopE was expressed mainly in the form of inclusion bodies.Western-blot result showed that the PcAbs could detect the SopE specifically.Moreover,the expression of SopE protein decreased in RyhB single deletion strain and even a significant decrease in RyhB double deletion strains compared with wild-type strain SE50336.It indicated that RyhB-1 and RyhB-2 upregulated the expression of SopE.Above results laid a foundation for further study on the study mechanism of RyhB-1 and RyhB-2 on SopE.