【摘要】 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。更多还原

【Abstract】 Objective To produce a secretary recombinant NS1 of dengue virus type 2 (DENV2) and prepare the anti-NS1 polyclonal antibody. Method The full-length NS1 gene was expressed with Pichia pastoris expression system and the recombinant NS-1 was used to immunize rabbit to prepare anti-NS1 polyclonal antibody. The antibody was detected by Western-blot and quantified by ELISA. The polyclonal antiserum was purified by caprylic acid-ammonium sulfate precipitation and protein A resin. The specificity and sensitivity of the antibody were determined by ELISA and Western-blot after purification. Result DENV2 NS1 protein was expressed efficiently in Pichia pastoris. The antibody could specifically recognize DENV2 NS1 protein and the titer of the antiserum obtained in this experiment was up to 1∶6000. Conclusion The successful preparation of the polyclonal antibody against NS1 protein provides an important reagent for further study of the role of NS1 in the pathogenesis of dengue virus.更多还原