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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 test its immunogenicity against mice. Methods Recombinant Escherichia coli BL21-pET30-EEEV-E2 was induced by IPTG to express E2 protein. The recombinant E2 protein was purified by inclusion body purification kit and analyzed by SDS-PAGE and Western blot. The BALB / c mice were randomly divided into 4 groups: PBS control group, Freund’s adjuvant control group (emulsified with Freund’s adjuvant and PBS at a volume ratio of 1: 1), E2 protein group : 1 mixture) and E2 protein + Freund’s adjuvant group (E2 protein and Freund’s adjuvant were emulsified in a volume ratio of 1: 1), and each group was immunized with muscle of mouse hindlimb three times for two times Interval time was 14 d, the immune dose was 100μl / only. On the 7th day after the second immunization, the proportion of CD4 + and CD8 + T cells in the mice was detected by flow cytometry. On the 14th day after the second immunization, the levels of IL-2 , IL-4 and IFNγ were detected. On the 7th day after the third immunization, the proliferation of mouse lymphocytes was detected by MTT assay. The serum IgG antibody titers were detected by ELISA method on the 14th day after each immunization. Results The expressed recombinant E2 protein had a relative molecular mass of about 53 000 and an expression level of 26.3% of the total bacterial proteins. The purity of the purified recombinant E2 protein was over 95%. The recombinant E2 protein expressed and purified could bind to the mouse anti-His tag Monoclonal antibody binding. Compared with the PBS control group, the Freund’s adjuvant control group and the E2 protein group, the ratio of CD4 + to CD8 + T cells in the E2 + Freund’s adjuvant group, the concentrations of IL-2, IL-4 and IFNγ in serum, (P <0.01). After the first immunization, mice could produce E2 protein IgG antibody. With the prolongation of immunization time, the antibody titers gradually increased. On the 14th day after the third immunization, , Antibody titers up to 1: 320. Conclusion The recombinant E2 protein was expressed and purified, which can produce a strong immune response in mice and provide a reference for the development of new EEEV vaccine.