LRP16基因的多克隆抗体制备及表达的探索

Antibody preparation and basic research of LRP16 gene in different tissues of human

目的:制备人LRP16蛋白多克隆抗体并利用该抗体检测LRP16蛋白表达及亚细胞定位。方法:利用已构建的pRSET-C载体和LRP16基因组成的连接产物转化BL-21缺陷型大肠杆菌。诱导该基因原核表达,快速蛋白免疫法获取相应的多克隆抗体,利用免疫组化法观察LRP16基因产物在人多种组织中的表达差异。结果:成功获得高效的LRP16抗体,利用该抗体初步证实LRP16基因编码产物的在不同雌激素受体水平的组织分布特点。结论:制备的LRP16抗体及其全长基因可用于LRP16基因的生理功能及病理学研究,初步发现LRP16基因的表达与雌激素受体呈正相关。

Objective： To prepare the anti-LRP16 polyclonal antibody and investigate the subcellular localization of LRP16 protein. Methods ：The full length LRP16 gene was obtained by RT-PCR.. The predicated antigen epitope was cloned into protein expression plasmid pRSET-C, transformed into E. coil BL21 （ DE3 ） cells for expressing recombinant pRSET-C-LRP16 protein induced by IPTG. The purified pRSET-C-LRP16 protein was employed to immunize rabbit for preparing the polyclonal antibody. The expression of LRP16 was analyzed by Western blot and immunohistochemistry in cancer such as ： breast cancer and ovarian serous cystadenoma. Results-Specific anti- LRP16 rabbit polyclonal antibody was obtained, and both Western blot and immunohistochemistry showed that LRP16 was expressed in the cancer with high-expressing of ER, localized in the cellular nucleus. Conclusions：LRP16 protein is a nuclear protein located in cellular nucleus. The anti LRP16 polyclonal antibody can be used for studying the physiological function of LRP16 and its pathology.