呈递预测的小鼠OX40胞外域抗原表位重组病毒样颗粒的制备

Preparation of recombinant virus-like particles presenting predicted antigenic epitopes of mouse OX40 extracellular domain

目的制备呈递预测的小鼠OX40胞外域抗原表位重组病毒样颗粒(virus-like particles,VLPs),为进一步以主动免疫方式靶向OX40调控T细胞免疫功能奠定基础。方法通过抗原数据库中氨基酸的出现频率,对肽段氨基酸组成进行分析,获得其作为抗原表位的潜能,从而预测潜在的OX40线性B细胞表位;经基因重组技术将抗原表位插入至乙型肝炎核心抗原(hepatitis B core antigen,HBcAg)的优势表位区域,在大肠埃希菌中表达,超声裂解菌体,上清中目的蛋白经硫酸铵沉淀及Sepharose 4 Fast Flow凝胶层析纯化,沉淀中目的蛋白经2%TritonX-100洗涤、尿素溶解及脱盐纯化,获得的蛋白经电镜观察VLPs形态。将VLPs经皮下免疫小鼠,ELISA法检测血清特异抗体水平。结果经分析预测获得7个潜在的抗原表位;嵌合表位的重组HBcAg蛋白(P1~P7)在大肠埃希菌中均获得了有效表达,其中P3、P6和P7蛋白存在于超声裂解菌体的可溶性上清中,而P1、P2、P4和P5蛋白存在于沉淀中。纯化的目的蛋白均以VLPs形式存在,免疫小鼠可有效激发OX40特异的抗体应答。结论成功构建并纯化制备了呈递OX40抗原表位的重组VLPs。

Objective To prepare recombinant virus-like particles(VLPs) presenting predicted antigenic epitopes of mouse OX40 extracellular domain and lay a foundation of regulating T cell function through targeting OX40 with an active immunization strategy. Methods Based on the statistical data of amino acid frequency in the known antigenic peptides,mouse OX40 was analyzed for potential linear B cell epitopes. The predicted epitopes were inserted into the immunodominant domain of hepatitis B core antigen(HBcAg) by gene recombinant technique,and expressed in E. coli. The bacterial cells were subjected to ultrasonic lysis,and the protein in the supernatant was purified by a procedure consists of ammonium sulfate precipitation and gel filtration chromatography,washed with TritonX-100,dissolved in urea,and desalted. The obtained proteins were observed for the morphology of VLPs by electron microscopy. Mice were immunized s. c. with the VLPs,of which the serum antibody level was determined by ELISA. Results Seven potential antigenic epitopes were obtained,and the seven corresponding recombinant proteins(P1 ~ P7)were successfully expressed,among which P3,P6 and P7 existed in the supernatants of ultrasonic lysates while P1,P2,P4 and P5 in the precipitate. All the purified proteins formed VLPs under electron microscopy,which elicited efficiently OX40-specific antibody response in mice. Conclusion Recombinant VLPs presenting OX40 antigenic epitopes were successfully constructed and purified.