铜绿假单胞菌重组PcrV-OprI疫苗保护小鼠骨折固定术后感染的作用

Recombinant protein vaccine PcrV-OprI prevents mice against Pseudomonas aeruginosa infection after intramedullary nailing

目的构建髓内针固定小鼠开放性股骨骨折后铜绿假单胞菌(Pseudomonas aeruginosa,PA)感染模型,评价候选PA疫苗重组PcrV-OprI的免疫保护效果。方法采用大肠杆菌表达PcrV-OprI蛋白,依次经亲和、疏水层析纯化得到目的蛋白。C57BL/6J小鼠在第0、14、21天免疫PcrV-OprI蛋白后,使用酶联免疫吸附法检测血清中特异性IgG抗体及亚型效价。末次免疫后14 d,进行髓内针固定小鼠开放性股骨骨折术。术后PAO1菌株感染,通过比较免疫组与对照组细菌定植、体质量、组织病理学和X线影像表现等差异,评价PcrV-OprI抗原保护效果。结果PcrV-OprI免疫组小鼠血清中特异性抗体水平显著升高(P<0.05),且以IgG1亚型为主;PcrV-OprI免疫组体质量减轻幅度显著低于对照组(P<0.05),PcrV-OprI免疫组软组织、骨内、植入物细菌数量显著低于对照组(P<0.05);PcrV-OprI免疫组炎症病理损伤小于对照组;在观察期结束时,PcrV-OprI免疫组骨折线清晰且逐渐闭合,呈愈合趋势而对照组骨折处未见明显愈合。结论PcrV-OprI免疫能有效引起小鼠体液免疫应答,在开放性骨折髓内针固定术后PA感染模型中具有显著保护效果。

Objective To establish a model of Pseudomonas aeruginosa(PA)infection in mice with open fractures after the femur with intramedullary nailing and to explore the immunoprotective effects of recombinant PcrV-OprI.Methods PcrV-OprI protein was expressed in E.coli,and then the target protein was purified by affinity chromatography and hydrophobic interaction chromatography.The C57BL/6J mice were immunized with PcrV-OprI protein on Day 0,14 and 21.After that,the titers of specific IgGs and their subtypes were determined by ELISA.In 14 d after the final vaccination,open femur fractures and intramedullary nailing were performed in mice,which was followed by a challenge with PAO1 strain.The protection of PcrV-OprI was assessed with bacterial colonization,body weight,histology,and X-ray imaging.Results The levels of specific antibodies were significantly higher in PcrV-OprI-immunized sera than that of the control group(P<0.05),and the obtained IgG1 was the predominating subtype.The loss of body weight of the immunized mice was significantly lower than that of the control group(P<0.05).In addition,a similiar trend was observed in the amount of colonized bacteria in the tissues,bones,and implants(P<0.05).Moreover,the histological observation suggested that bacteria caused less damage in the PcrV-OprI-immunized mice.At the end of the observation period,the fracture line in immunized mice was clear,indicating gradual healing.Conclusion Vaccination of PcrV-OprI is efficient in causing a humoral immune response and shows significant protective effect on PA infection model in mice after open fractures of the femur with intramedullary nailing.