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目的构建登革2型病毒(dengue virus serotype2,DV2)非结构蛋白NS2B的真核表达质粒pCAGGS-P7/NS2B,进行DNA免疫,制备小鼠抗NS2B特异性多克隆抗体。方法PCR扩增DV2-NS2B基因片段,构建真核表达载体pCAGGS-P7/NS2B,采用脂质体介导的方法转染Vero细胞,检测目标蛋白的表达。用该表达质粒免疫BALB/c小鼠,制备抗NS2B抗血清。结果成功构建真核表达重组质粒pCAGGS-P7/NS2B,该重组质粒具有良好的免疫原性,免疫小鼠后所获的抗血清,抗体效价达1∶3200,Western blot和间接免疫荧光证实抗血清能特异性地识别DV2-NS2B蛋白。结论通过DNA免疫所制备小鼠抗DV2-NS2B抗血清能特异性地识别DV2-NS2B蛋白。
Objective To construct eukaryotic expression plasmid pCAGGS-P7 / NS2B of non-structural protein NS2B of dengue virus serotype2 (DV2) for DNA immunization and to prepare mouse anti-NS2B specific polyclonal antibody. Methods The DV2-NS2B gene fragment was amplified by PCR. The eukaryotic expression vector pCAGGS-P7 / NS2B was constructed and transfected into Vero cells by liposome-mediated method to detect the expression of target protein. BALB / c mice were immunized with this expression plasmid to prepare anti-NS2B antiserum. Results The eukaryotic expression plasmid pCAGGS-P7 / NS2B was constructed successfully. The recombinant plasmid has good immunogenicity. The antiserum obtained from mice immunized with the titer of antibody reached 1:3200. Western blot and indirect immunofluorescence confirmed the resistance Serum specifically recognizes DV2-NS2B protein. Conclusion The mouse anti-DV2-NS2B antiserum prepared by DNA immunization can specifically recognize DV2-NS2B protein.