异常凝血酶DCP单克隆抗体的制备及鉴定
发布时间：2019-05-08 15:40
【摘要】：目的 肝细胞性肝癌(Hepatocellular carcinoma, HCC)是最常见的恶性肿瘤之一,其发病率和死亡率呈逐年上升的趋势,早期诊断和早期治疗对肝癌病人的预后起着决定性的作用。目前HCC的常规筛查和诊断方法包括体格检查、影像学检查及血清甲胎蛋白(Alpha fetoprotein, AFP)检测。AFP虽为诊断HCC的首选指标,但其敏感性只有60%-70%,特别是早期HCC, AFP敏感性仅40%左右。最近的研究发现,血清异常凝血酶DCP (des-γ-carboxy prothrombin, DCP)在诊断HCC中其敏感性和特异性是可以与AFP相媲美的一个标志物。因此,本试验拟制备抗DCP单克隆抗体(mAb),初步鉴定其生物学功能,并为进一步将所制备的单克隆抗体用于建立定量检测DCP的方法奠定基础。 方法 1.构建DCP原核表达载体pCold Ⅱ-DCP,导入E.coli. BL21(DE3)菌株,IPTG诱导表达后进行SDS-PAGE电泳鉴定,以His TrapFF亲和层析柱纯化His-DCP融合蛋白。 2.以该融合蛋白免疫BALB/c小鼠,取免疫后小鼠的脾细胞与骨髓瘤细胞Sp2/0融合,依次进行阳性杂交瘤细胞株的筛选及亚克隆化,以获得能稳定分泌a-抗DCP mAb杂交瘤单克隆细胞株。 3.以间接ELISA法检测抗体效价；以Sigma公司提供的mAb亚类检测试剂盒进行抗体亚类鉴定；以Western blot及免疫组织化学鉴定抗体特异性。 结果 1.成功构建了表达质粒pCold Ⅱ-DCP。 2.成功诱导表达并纯化了His-DCP融合蛋白,浓度为1.5mg/mL;纯度达80%以上。 3.获得了2株稳定分泌抗人DCP mAb杂交瘤细胞株,分别命名为385和6H4；其免疫球蛋白亚类都为IgG2a型,每株抗体效价均达1：243,000以上。利用Western blot和免疫组织化学结果显示抗人DCP单克隆抗体能与DCP发生特异性结合。 结论 本研究成功构建了pCold Ⅱ-DCP表达质粒,并表达了His-DCP融合蛋白；以该融合蛋白为免疫原,成功制备出了抗人DCP mAb,为进一步将该抗体应用于肝癌的早期诊断提供了重要工具。
[Abstract]:Aim: hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is one of the most common malignant tumors. Its morbidity and mortality are increasing year by year. Early diagnosis and early treatment play a decisive role in the prognosis of HCC patients. At present, the routine screening and diagnosis methods of HCC include physical examination, imaging examination and serum alpha-fetoprotein (Alpha fetoprotein, AFP) detection. Although AFP is the first choice for the diagnosis of HCC, its sensitivity is only 60% ~ 70%, especially in the early stage of HCC,. The sensitivity of AFP was only about 40%. Recent studies have found that the sensitivity and specificity of serum abnormal thrombin DCP (des- 纬-carboxy prothrombin, DCP) in the diagnosis of HCC is comparable to that of AFP. Therefore, the anti-DCP monoclonal antibody (mAb), was prepared to identify its biological function and lay a foundation for the further application of the monoclonal antibody to establish a quantitative method for the detection of DCP. Method 1. The prokaryotic expression vector pCold 鈪，
本文编号：2472023
[Abstract]:Aim: hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is one of the most common malignant tumors. Its morbidity and mortality are increasing year by year. Early diagnosis and early treatment play a decisive role in the prognosis of HCC patients. At present, the routine screening and diagnosis methods of HCC include physical examination, imaging examination and serum alpha-fetoprotein (Alpha fetoprotein, AFP) detection. Although AFP is the first choice for the diagnosis of HCC, its sensitivity is only 60% ~ 70%, especially in the early stage of HCC,. The sensitivity of AFP was only about 40%. Recent studies have found that the sensitivity and specificity of serum abnormal thrombin DCP (des- 纬-carboxy prothrombin, DCP) in the diagnosis of HCC is comparable to that of AFP. Therefore, the anti-DCP monoclonal antibody (mAb), was prepared to identify its biological function and lay a foundation for the further application of the monoclonal antibody to establish a quantitative method for the detection of DCP. Method 1. The prokaryotic expression vector pCold 鈪，
本文编号：2472023
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