HIV-1 p15(Gag)基因的克隆、蛋白表达及抗体制备

Cloning,expression,and antibody preparation of HIV-1 p15(Gag)

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 prepare HIV-1 p15（Gag） protein and its antibody for studying the biological activity of HIV-1p15（Gag）. Methods The full length gene fragment of p15（Gag） was amplified by PCR, and then cloned into pET28a（ ＋ ） prokaryotic expressing vector. pET28a（ ＋ ）-p15（Gag） was induced by IPTG in E. coli BL21 and the expressed protein was detected by Western blot assay. The purified protein was used to immune rabbits for preparing polyclonal antibody. The specificity and titer of the antibody were identified with ELISA and immunohistochemistry, respectively. Results The plasmid pET28-p15（Gag） was successfully constructed and HIV-1 p15（Gag） protein could be expressed in E. coli BL21 with high efficiency. The recombinant protein was characterized with anti-his and HIV-1 positive plasma by Western blot assay. Rabbit immunized with the purified protein produced high titer antibody. The viral protein expressed in eukaryotic cells was specifically combined with the antibody detected by immunohistochenaistry. Conclusion Purified protein HIV-1 p15 with high antigenicity is obtained. Prepared polyclonal antibody can be used to detect the viral protein that expressed in eukaryotic cells, which provides rehable tools for the future HIV-1 research.