HIV-1 p15（Gag）基因的克隆、蛋白表达及抗体制备

Expression Cloning and Antibody Preparation of HIV - 1 p15 Gag

目的：构建HIV-1p15（Gag）原核表达载体，诱导蛋白表达及制备多克隆抗体。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，利用大肠杆菌表达系统表达HIV-1p15蛋白，分别用His抗体和HIV阳性血清做westernblot鉴定目的蛋白的免疫原性。纯化目的蛋白免疫家兔，制备多克隆抗体。通过酶联免疫吸附实验（ELISA），细胞免疫组织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BL2。（DE3）中用IPTG诱导表达，得到相对分子质量约20KD的p15（Gag）蛋白，经westernblot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 construct expression vector HIV - lp15 Gag and prepared its antibody. Methods： The full length gene fragment of p15 Gag was amplified by PCR. Then it was cloned into pET28a（ ＋ ） prokaryotic expressing vector, pET28a（ ＋ ） - p15 Gag was induced by IPTG in E. coil BL21 （DE3） and the expressed protein was detected by Western Blot. The purified protein was used to immunize rabbits to prepare polyclonal antibody. Rabbit serum specificity and its titer were assyed by ELISA and cell immunohistochemistry. Results： We constructed pET28 - pl5 Gag successfully. HIV - 1 p15 Gag protein can be expressed in E. coli BL21 （DE3） with high efficiency, it was a kind of 20KD molecular weight protein with His - tag. The ratio of protein to total bacteria protein was 23%. We got corresponding antibody through immunizing rabbit with the purified protein. The data of Western blot and ELISA demonstrated that the purified antigen had high antigenicity. It can react with positive serum of HIV - 1 patient specifically. The rabbit serum can combine with purified protein and it had a high titer, cell immunohistochemistry result shows that the antibody can combine with virus protein that expressed in eukaryotic cells. Conclusions： We obtained purified protein HIV - 1 p15, it had high antigenicity. Prepared polyclonal antibody can detect virus protein that expressed in eukaryotic cell, which provided reliable tools for the future research.