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目的获取H5N1亚型禽流感病毒NS1蛋白的多克隆抗血清,制备NS1蛋白的多克隆抗体。方法利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达融合蛋白GST-NS1,采用Glutathione-sepharose 4B亲和层析纯化融合蛋白。纯化的GST-NS1蛋白以不同剂量皮下注射免疫BALB/c小鼠,收集抗血清,采用Protein A-sepharose和CNBr-sepharose 4B亲和层析从抗血清中纯化抗NS1蛋白的多克隆抗体。Western blot和免疫荧光法鉴定多克隆抗体的特异性,酶联免疫吸附法(ELISA)检测抗体的效价。结果 SDS-PAGE结果表明,融合蛋白GST-NS1以可溶形式表达,诱导4 h的表达量占细菌可溶性蛋白的32.6%,融合蛋白的纯度达到90%以上。Western blot分析显示,亲和层析制备的多克隆抗体对NS1蛋白具有高度特异性。ELISA结果表明,皮下注射25、50、100μg GST-NS1融合蛋白的BALB/c小鼠血清光密度D(450)值均明显增高(P<0.01),其滴度达到1∶3 200。免疫荧光观察到病毒感染的A549细胞中,有内源性NS1蛋白的表达。结论成功地从抗血清中制备出NS1蛋白的多克隆抗体,制备的抗体具有高度特异性。
Objective To obtain polyclonal antiserum against NS1 protein of H5N1 avian influenza virus and prepare polyclonal antibody against NS1 protein. Methods The fusion protein GST-NS1 was induced by isopropyl-β-D-thiogalactoside (IPTG), and the fusion protein was purified by Glutathione-sepharose 4B affinity chromatography. BALB / c mice were immunized subcutaneously with different doses of purified GST-NS1 protein. Antiserum was collected and polyclonal antibodies against NS1 protein were purified from antisera using Protein A-sepharose and CNBr-sepharose 4B affinity chromatography. The specificity of polyclonal antibody was identified by Western blot and immunofluorescence assay. The antibody titer was detected by enzyme-linked immunosorbent assay (ELISA). Results The results of SDS-PAGE showed that the fusion protein GST-NS1 was expressed in soluble form. The expression of GST-NS1 reached 32.6% after 4 h of induction and the purity of the fusion protein was over 90%. Western blot analysis showed that polyclonal antibodies prepared by affinity chromatography were highly specific to NS1 protein. The results of ELISA showed that the serum optical density D (450) of BALB / c mice injected with 25, 50 and 100μg of GST-NS1 fusion protein were significantly increased (P <0.01) with a titer of 1: 300. Immunofluorescence showed the expression of endogenous NS1 protein in virus-infected A549 cells. Conclusion The polyclonal antibody of NS1 protein was successfully prepared from antiserum. The prepared antibody was highly specific.