东方马脑炎病毒E2蛋白的表达、纯化及免疫原性

Expression,purification and immunogenicity of E2 protein of eastern equine encephalitis virus

目的表达、纯化东方马脑炎病毒(eastern equine encephalitis virus,EEEV)E2蛋白,并检测其对小鼠的免疫原性。方法利用IPTG诱导重组大肠埃希菌BL21-pET30-EEEV-E2,表达E2蛋白,用包涵体纯化试剂盒纯化重组E2蛋白,进行SDS-PAGE和Western blot分析。将BALB/c小鼠随机分为4组:PBS对照组、弗氏佐剂对照组(弗氏佐剂与PBS按体积比1∶1乳化)、E2蛋白组(E2蛋白与PBS按体积比1∶1混合)和E2蛋白+弗氏佐剂组(E2蛋白与弗氏佐剂按体积比1∶1乳化),每组10只,各组均经小鼠后肢肌肉免疫3次,两次免疫间隔时间均为14 d,免疫剂量均为100μl/只。第2次免疫后第7天,采用流式细胞术检测小鼠体内CD4+和CD8+T细胞比例;第2次免疫后第14天,采用细胞因子ELISA定量试剂盒检测小鼠血清中IL-2、IL-4和IFNγ的含量;第3次免疫后第7天,采用MTT法检测小鼠淋巴细胞增殖情况;每次免疫后第14天,采用ELISA法检测小鼠血清中IgG抗体效价。结果表达的重组E2蛋白相对分子质量约为53 000,表达量为菌体总蛋白的26.3%;纯化的重组E2蛋白纯度达95%以上;表达和纯化的重组E2蛋白均可与鼠抗His标签单克隆抗体结合。与PBS对照组、弗氏佐剂对照组和E2蛋白组相比,E2蛋白+弗氏佐剂组小鼠体内CD4+与CD8+T细胞比值、血清中IL-2、IL-4和IFNγ浓度、体内淋巴细胞增殖指数均明显升高(P﹤0.01);小鼠初次免疫后即可产生E2蛋白IgG抗体,且随着免疫时间的延长,抗体效价逐渐上升,第3次免疫后第14天,抗体效价可达1∶320。结论表达并纯化了重组E2蛋白,其能使小鼠产生较强的免疫反应,为新型EEEV疫苗的研制提供了参考。

Objective To express and purify the E2 protein of eastern equine encephalitis virus（EEEV） and determine its immunogenicity in mice.Methods Recombinant E.coi BL21-pET30-EEEV-E2 was induced with IPTG,and the expressed E2 protein was purified by inclusion body purification kit and identified by SDS-PAGE and Western blot.BALB / c mice were randomly divided into PBS control,Freund adjuvant control（Freund adjuvant emulsified with PBS at a volume ratio of 1 ∶ 1）,E2 protein（mixture of E2 protein and PBS at a volume ratio of 1 ∶ 1） and E2 protein ＋ Freund adjuvant（E2 protein emulsified with Freund adjuvant at a volume ratio of 1 ∶ 1） groups,10 for each.The mice in various groups were immunized i.m.for 3 times each at a dosage of 100 μl and an interval of 14 d.The ratio of CD4＋ to CD8＋ T cell was detected by flow cytometer on day 7,while the IL-2,IL-4 and IFNγ in sera by quantitative ELISA kit on day 14,after the second immunization.The proliferation of lymphocytes was determined by MTT method on day 7 after the third immunization,while the IgG titer in sera by ELISA on day 14 after each immunization.Results The expressed recombinant E2 protein,with a relative molecular mass of 53 000,contained 26.3% of total somatic protein and reached a purity of more than 95% after purification.Both the expressed and purified recombinant E2 protein showed specific binding to mouse monoclonal antibody against His tag.The ratio of CD4＋ T to CD8＋ T cells,concentrations of IL-2,IL-4 and IFNγ,and lymphocyte proliferation index of mice in E2 protein ＋ Freund adjuvant group were significantly higher than those in PBS control,Freund adjuvant control and E2 protein groups（P 0.01）.IgG antibody titer against E2 protein was induced in mice after the first immunization,and increased gradually with the increasing time,which reached 1 ∶ 320 on day 14 after the third immunization.Conclusion Recombinant E2 protein was expressed and purified,which induced strong immune response in mice and provided a reference for development of novel EEEV vaccine.