Notch信号途径效应分子HES1的原核表达及其多克隆抗体的制备与鉴定

Prokaryotic expression of HES1 and preparation of its polyclonal antibody

目的 原核表达HES1分子 ,并制备其特异性多克隆抗体 ,以研究该分子在肿瘤细胞中的表达情况。方法 诱导表达GST HES1融合蛋白和GST ,经谷胱甘肽 Sepharose 4B亲和纯化后 ,用GST偶联预激活的Sepharose 4B ,以GST HES1为免疫原制备兔抗血清。得到的抗血清经GST Sepharose 4B吸收抗GST成分 ,以间接ELISA和Westernblot鉴定抗体特异性 ,以Westernblot分析胃癌、结肠癌及其相应非癌变组织中HES1的表达水平。结果 成功地表达并纯化了GST HES1融合蛋白。制备的抗HES1多克隆抗体具有很高的特异性 ,与GST或大肠杆菌成分无交叉反应 ,用于进行天然HES1分子的Westernblot检测时效果良好。初步检测发现 ,胃癌和结肠癌中HES1的表达水平明显高于相应的正常组织。结论 通过原核表达并纯化HES1,成功地制备了该分子的特异性多克隆抗体 。

Aim To express HES1 and prepare its specific antibodies for investigation of its expression in tumors. Methods GST HES1 fusion protein and GST were induced to express in E. Coli , then were purified with glutathione Sepharose 4B. Purified GST HES1 was used to immunize rabbit and purified GST was coupled with CNBr activated Sepharose 4B. The rabbit anti HES1 serum was harvested and the anti GST antibodies in the serum were absorbed with GST Sepharose 4B. The specificity of the ployclonal anti HES1 antibody was determined by indirect ELISA and Western blot. The expression of HES1 in gastric cancer, colon cancer and corresponding non tumor tissues were detected by Western blot. Results GST HES1 fusion protein was expressed and purified successfully. Prepared ployclonal anti HES1 antibody could recognize native HES1 and showed high specificity against HES1 without cross reaction with GST or E. Coli component in ELISA and Western blot. Preliminary western blot result indicated that the expression of HES1 in gastric cancer and colon cancer was significantly higher than that in corresponding non tumor tissues. Conclusion HES1 successfully expressed in E.coli and purified. Bloyclonal anti HES1 antibody with high specificity is prepared and applied successfully in preliminary investigation.