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目的为了研究HIV-1蛋白酶的生物学活性,制备HIV-1 PR蛋白及其特异性抗体。方法用PCR方法扩增编码PR的基因序列,将其克隆到原核表达载体pET28a(+)中,并表达HIV-1 PR蛋白,用His抗体为一抗做Western blot鉴定目的蛋白。以纯化的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pET28 a(+)-PR成功构建,并可在大肠杆菌BL21(DE3)中诱导表达,得到的PR蛋白经SDS-PAGE和Western blot鉴定正确。用纯化蛋白免疫家兔,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-1PR蛋白,制备的多克隆抗体能够检测自然状态下病毒蛋白PR,为进一步研究HIV-1奠定了实验基础。
Objective To investigate the biological activity of HIV-1 protease, HIV-1 PR protein and its specific antibodies were prepared. Methods The gene encoding PR was amplified by PCR and cloned into prokaryotic expression vector pET28a (+). The recombinant protein was expressed by Western blot using His antibody as primary antibody. The purified target protein was used as antigen 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 pET28 a (+) - PR was successfully constructed and expressed in E. coli BL21 (DE3). The obtained PR protein was identified by SDS-PAGE and Western blot. Rabbit immunized with purified protein, the prepared polyclonal antibody has strong immunospecific. Conclusions The purified HIV-1 PR protein was obtained. The prepared polyclonal antibody could detect the viral protein PR in its natural state, which laid the experimental foundation for the further study of HIV-1.