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目的分泌表达重组登革病毒2型NS1蛋白,并制备兔抗NS1多克隆抗体。方法应用毕赤酵母表达系统表达全长登革病毒2型NS1蛋白,制备抗原,免疫家兔;采集免疫血清,制备兔抗NS1蛋白多克隆抗体;应用Western-blot、ELISA法鉴定和检测抗体效价;经辛酸-硫酸铵法、亲和层析法纯化抗体,SDS-PAGE电泳检测抗体的纯度;用Western-blot、ELISA法检测纯化后IgG性质及效价。结果重组NS1蛋白获得分泌表达,其免疫血清经Western-blot、ELISA法证实获得特异性兔抗NS1多克隆抗体,抗体效价为1∶6000。结论重组登革病毒2型NS1蛋白在毕赤酵母真核表达系统中高效表达,纯化产物有较强的免疫原性,成功获得特异性兔抗NS1多克隆抗体,为进一步研究登革病毒NS1蛋白及其抗体在登革病毒致病与免疫机制中的作用奠定了基础。
Objective To express recombinant dengue virus type 2 NS1 protein and prepare rabbit anti - NS1 polyclonal antibody. Methods The full-length Dengue virus type 2 NS1 protein was expressed by using Pichia pastoris expression system to prepare antigens for immunization of rabbits. The serum was collected to prepare polyclonal antibody against NS1 protein. Western blotting and ELISA were used to identify and test the antibody efficacy Antibodies were purified by octanoic acid-ammonium sulfate method and affinity chromatography. The purity of the antibody was detected by SDS-PAGE electrophoresis. The purified IgG was characterized by Western-blot and ELISA. Results The recombinant NS1 protein was secreted and expressed. The immune serum was confirmed by Western-blot and ELISA. The specific antibody titer was 1: 6000. Conclusion The recombinant dengue virus type 2 NS1 protein is highly expressed in the eukaryotic expression system of Pichia pastoris. The purified product has strong immunogenicity and successfully obtained the specific rabbit anti-NS1 polyclonal antibody. In order to further study the dengue virus NS1 protein And its antibody in the pathogenesis of dengue virus and immune mechanism laid the foundation.