UROC28基因的原核表达及抗UROC28多抗制备和初步应用

目的: 在原核细胞大肠杆菌中融合表达UROC28基因产物,分离、纯化后制备其特异性多克隆抗体,并初步应用于肿瘤标本的检测,为进一步研究该分子的功能和组织分布等提供条件. 方法: 利用DNA重组技术将UROC28基因克隆入融合表达载体pRSET-c质粒,并在大肠杆菌BL-21中融合表达目的蛋白,回收后制备兔抗血清,Western blot鉴定抗体后,应用该抗体对前列腺癌石蜡切片进行免疫组化染色. 结果: His-UROC28融合蛋白的Mr约为22 000,与预期相符;融合蛋白占菌体总蛋白的20%. 目的蛋白免疫新西兰兔获得多克隆抗体,Western blot结果显示制备的抗UROC28多克隆抗体具有较高的特异性,无明显交叉反应,并成功在前列腺癌组织标本中检测UROC28的表达. 结论: 本研究成功构建了UROC28融合表达载体,并在大肠杆菌中获得高效表达. 制备了该分子的多克隆抗体,经初步验证其效价高、特异性好,为深入研究UROC28的功能及其与疾病的关系创造了条件.

Prokaryotic expression of human UROC28 gene and preparation of its polyclonal antibody

AIM: To express human UROC 28 gene and to prepare its specific polyclonal antibodies for the diagnosis and treatment of prostate cancer. METHODS: UROC 28 gene was cloned into the expression vector pRSET c and His UROC28 fusion protein was expressed by IPTG induction and purified with SDS PAGE from the total proteins of E.coli BL 21 (DE3). New Zealand rabbits were immunized with this protein to prepare polyclonal antiserum and the specificity of the polyclonal anti UROC 28 antibody was determined by Western blot. RESULTS: His UROC28 fusion protein ( M r 22 000) was successfully expressed. Western blot showed that the polyclonal anti UROC 28 antibody recognize native UROC28 and was of high specificity against UROC28 without cross reaction. The expression of UROC28 was also successfully detected in prostate cancer tissues. CONCLUSION: UROC28 has been successfully expressed in BL 21. Polyclonal anti UROC 28 antibody with high specificity has been prepared and applied successfully as a tumor marker.