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目的:为研究HIV-1 NCp15的生物学活性,制备HIV-1 NCp15蛋白及其鸡卵黄抗体。方法:用PCR的方法扩增编码NCp15基因序列,将其克隆到原核表达载体PGEX4T1中表达HIV-1 NCp15蛋白,用HIV阳性血清做Western blot鉴定目的蛋白。以纯化的NCp15蛋白抗原免疫产蛋鸡,制备卵黄抗体。通过酶联免疫吸附实验(ELISA),Western blot检测抗体滴度及特异性。结果:成功构建原核表达载体PGEX4T1-NCp15,并可在大肠杆菌BL21(DE3)中诱导表达,得到相对分子质量约45ka的NCp15融合蛋白,经Western blot鉴定正确。纯化蛋白免疫产蛋鸡,制备的鸡卵黄抗体IgY具有较强免疫特异性。结论:得到纯化的HIV-1NCp15蛋白,制备的IgY能够检测HIV-1病毒蛋白NCp15,为进一步研究HIV-1奠定了实验基础。
AIM: To investigate the biological activity of HIV-1 NCp15, HIV-1 NCp15 protein and its chicken egg yolk antibody were prepared. Methods: The NCp15 gene sequence was amplified by PCR and cloned into the prokaryotic expression vector PGEX4T1 to express HIV-1 NCp15 protein. The HIV-1 positive serum was used to identify the target protein. Egg laying hens were immunized with purified NCp15 protein antigen to prepare egg yolk antibody. Antibody titer and specificity were determined by ELISA and Western blot. Results: The prokaryotic expression vector PGEX4T1-NCp15 was successfully constructed and expressed in E. coli BL21 (DE3). The NCp15 fusion protein with relative molecular mass of about 45ka was obtained and identified by Western blot. Purified protein immunization of laying hens, the prepared egg yolk antibody IgY has strong immunospecific. CONCLUSION: The purified HIV-1NCp15 protein was obtained. The prepared IgY could detect HIV-1 virus protein NCp15, which laid the foundation for further study on HIV-1.