HIV-1蛋白酶基因的克隆、蛋白表达及抗体制备

Expression of HIV-1 recombinant protease and its antibody preparation

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。

To prepare HIV-1 protease protein （PR） and its antibody for studying the biological activity of HIV-1 PR. The full length gene fragment of HIV-1 PR was amplified by PCR, and then cloned into pET28a （＋） prokaryotie expressing vector; the recombinant protein PR was induced by IPTG in E. colt BL21 and the expressed protein was detected by Western blot assay. Subsequently, the purified protein was used to immune rabbits for preparing polyclonal antibody, and then the specificity and titer of the antibody were detected respectively by ELISA and immunohistochemistry. We constructed the plasmid pET28a （＋） -PR successfully and expressed HIV-1 PR protein in E. colt BL21 efficiently; the recombinant protein was characterized with His antibody by Western blot analyzing, while the rabbit immunized with the purified protein produced high titer of antibody. The viral protein expressed in eukaryotic cells was specifically combined with the antibody by immunohistochemistry. All results showed that the expressed recombinant HIV-1 PR possessed high antigenicity, and the prepared polyclonal antibody could be used to detect HIV-1 viral protein that expressed in eukaryotic cells, which provided reliable tools for the future HIV-1 research.