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目的为了研究HIV-1p15(Gag)的生物学活性,制备HIV-1p15(Gag)蛋白及其特异性抗体。方法用PCR的方法扩增编码p15(Gag)基因序列,将其克隆到原核表达载体pET28a(+)中表达HIV-1p15蛋白,分别用His抗体和HIV阳性血清做western blot鉴定目的蛋白。以纯化目的蛋白为抗原免疫日本大耳白兔,制备多克隆抗体。通过酶联免疫吸附实验(ELISA),免疫细胞化学法检测抗体滴度及其特异性。结果原核表达载体pET28a(+)-p15(Gag)成功构建,可在大肠杆菌BL21(DE3)中诱导表达,得到相对分子质量约20000的p15(Gag)蛋白经western blot鉴定正确。纯化蛋白免疫家兔,制备的多克隆抗体具有较强免疫特异性。结论得到纯化的HIV-1p15蛋白,制备的多克隆抗体能够检测自然状态下病毒蛋白p15(Gag),为进一步研究HIV-1奠定了实验基础。
Objective To investigate the biological activity of HIV-1p15 (Gag), HIV-1p15 (Gag) protein and its specific antibodies were prepared. Methods The sequence of p15 (Gag) gene was amplified by PCR and cloned into prokaryotic expression vector pET28a (+) for expression of HIV-1p15 protein. Western blot was used to identify the target protein with His and HIV positive sera, respectively. The purified protein of interest was used to immunize Japanese white rabbits to prepare polyclonal antibodies. Antibody titers and their specificity were determined by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Results The prokaryotic expression vector pET28a (+) - p15 (Gag) was successfully constructed and expressed in E. coli BL21 (DE3). The p15 (Gag) protein with the relative molecular mass of about 20,000 was identified by western blot. The purified protein was used to immunize rabbits and the prepared polyclonal antibody was more immunospecific. Conclusion The purified HIV-1p15 protein was obtained. The prepared polyclonal antibody can detect the natural protein p15 (Gag), which lays the foundation for the further study of HIV-1.