铜绿假单胞菌表面(氧化)低密度脂蛋白配体的研究

Detection of (oxidized) low-density lipoprotein binding ligand on Pseudomonas aeruginosa

摘要:

【背景】研究发现铜绿假单胞菌(Pseudomonas aeruginosa)与(氧化)低密度脂蛋白(low density lipoprotein，LDL/oxidized low density lipoprotein，oxLDL)具有特异性相互作用，有报道证实P. aeruginosa表达的RahU蛋白可以与LDL/oxLDL特异性结合。【目的】验证RahU蛋白是否是P. aeruginosa表面主要的LDL/oxLDL配体。【方法】大肠杆菌表达RahU蛋白(rRahU)，ELISA验证rRahU与LDL/oxLDL的相互作用。利用同源重组的方法构建RahU基因缺失突变株(ΔRahU菌株)作为阴性对照菌株，制备小鼠抗rRahU抗体，经Western Blot及ELISA分别检测抗rRahU抗体与P. aeruginosa野生型菌株膜蛋白中RahU蛋白及菌体表面RahU蛋白的结合。通过ELISA方法对P. aeruginosa野生型菌株及ΔRahU菌株与LDL/oxLDL的结合差异进行比较，并对不同蛋白酶水解ΔRahU菌体表面蛋白后ΔRahU菌株与LDL/oxLDL结合能力的差异进行比较。【结果】经ELISA验证rRahU与LDL/oxLDL存在特异性结合。Western Blot及ELISA方法证实小鼠抗rRahU抗体可以与P. aeruginosa野生型菌株膜蛋白中RahU蛋白及菌体表面RahU蛋白特异性结合，而不与ΔRahU菌株相互作用。P. aeruginosa野生型菌株及ΔRahU菌株与LDL/oxLDL结合能力无显著差异，且蛋白酶水解后ΔRahU菌株与LDL/oxLDL的结合能力相近。【结论】RahU蛋白是P. aeruginosa表面的LDL/oxLDL配体之一，但不是唯一的配体。

Abstract:

[Background] Pseudomonas aeruginosa (P. aeruginosa) specifically interacts with (oxidized) low density lipoprotein (LDL/oxLDL), and RahU expressed by P. aeruginosa binds to LDL/oxLDL. [Objective] Our study investigated if RahU on P. aeruginosa was the major binding ligand for LDL/oxLDL. [Methods] The interaction of recombinant RahU (rRahU) with LDL/oxLDL was detected by ELISA. Mouse anti-rRahU antibody was generated to determine if RahU was expressed on the membrane of the wild P. aeruginosa strain by Western-Blotting and ELISA. The affinity of LDL/oxLDL bind to wild P. aeruginosa strain and ΔRahU strain (constructed as a negative control) was compared by ELISA. Influence of different protease digestion on the binding affinity of ΔRahU strain to LDL/oxLDL was also compared by ELISA. [Results] Recombinant RahU specifically interacted with LDL/oxLDL. Western-Blotting and ELISA results revealed that anti-rRahU antibody bound to the membrane protein extracted from wild P. aeruginosa strain specifically, but not with ΔRahU strain. Binding affinity of LDL/oxLDL with both wild P. aeruginosa strain and ΔRahU strain made no significant difference. Protease digestion did not influence the binding of ΔRahU strain to LDL/oxLDL. [Conclusion] RahU is one but not the unique ligand of LDL/oxLDL on P. aeruginosa.