人APE1单克隆抗体的制备及其在肿瘤血清学检测中的应用研究

发布时间：2018-04-27 22:42

  本文选题：肿瘤标志物 + 脱嘌呤/脱嘧啶核酸内切酶　； 参考：《第三军医大学》2009年硕士论文

【摘要】： 肿瘤的早期发现和诊断对肿瘤的预防和治疗至关重要。肿瘤标志物作为肿瘤临床诊断的常规手段之一,对肿瘤的早期发现和疗效观察具有重要意义。随着ELISA、RT-PCR、FISH、SELDI-TOF、蛋白质组学、组织芯片和基因芯片等生物技术的飞速发展,使得许多具有应用前景的肿瘤标志物逐一被发现,但目前仍无任何一种肿瘤标志物可对恶性肿瘤进行准确诊断和预后预测,肿瘤的实验室诊断尚缺乏高敏感性和高特异性的检测方法。研究表明,现有肿瘤标志物存在特异性和阳性率低的缺点,不能很好达到肿瘤“早期诊断”这一目的。对肿瘤标志物进行联合检测能提高恶性肿瘤诊断的灵敏度及特异度,但仍存在诊断总准确率不高的缺陷。因此,寻找一种新的高敏感、高特异性的肿瘤标志物或建立一种新的肿瘤标志物的联合检测方法成为大多数实验室研究的方向之一。 人脱嘌呤脱嘧啶核酸内切酶(Human apurinic/apyrimidinic endonuclease/redox effector factor,hAPE1)是DNA损伤碱基切除修复(base excision repair, BER)途径的关键酶,是一种多功能蛋白质,具有修复AP位点和氧化还原双重功能,与细胞凋亡,肿瘤细胞的增殖、分化和转化,放化疗敏感性以及神经系统退行性变化有着密切的关系。国内外研究表明,hAPE1蛋白在机体各器官组织中均有表达,而肿瘤组织中的hAPE1定位和表达与相应的正常组织有显著差异,其表达水平与肿瘤放化疗抵抗有关。本课题前期研究发现,肿瘤患者血清中hAPE1蛋白表达显著高于正常人。因此,hAPE1的表达水平(和/或类型)可作为筛选某些肿瘤的辅助手段,hAPE1有望成为肿瘤早期诊断中一项敏感而特异的指标。我们推测,对hAPE1进行血清学检测可能有助于恶性肿瘤的早期临床诊断,并有效判断肿瘤细胞放疗和化疗的敏感性。 研究目的 1.构建hAPE1原核表达载体,诱导表达、纯化hAPE1融合蛋白,并对其生物学功能进行初步分析; 2.建立稳定分泌hAPE1单克隆抗体的杂交瘤细胞株,制备并纯化hAPE1单克隆抗体,鉴定其生物学特性并进行初步应用; 3.初步应用hAPE1血清学检测方法探讨hAPE1对恶性肿瘤的诊断作用,并与多肿瘤标志物蛋白芯片检测方法比较,探讨hAPE1和多肿瘤标志物蛋白芯片联合检测在恶性肿瘤诊断中的临床意义。 研究内容和方法 1. hAPE1原核表达载体的构建、融合蛋白的纯化及功能鉴定 通过分子克隆技术构建包含hAPE1全长基因序列的pET28a-hAPE1原核表达载体,经测序验证后,将pET28a-hAPE1重组质粒转化入E.coli BL21(DE3),IPTG诱导表达并鉴定,以His亲和层析技术纯化蛋白,用Western blot及EMSA实验分析hAPE1融合蛋白的生物学功能。 2. hAPE1单克隆抗体的制备、生物学特性鉴定及初步应用 以纯化的hAPE1融合蛋白为抗原四肢皮下和腹腔注射免疫Balb/c小鼠,应用细胞融合、间接ELISA法筛选和克隆化技术获得稳定分泌hAPE1单克隆抗体的杂交瘤细胞株。以小鼠体内诱生法产生腹水,鉴定单抗亚类后采用Protein G柱对其纯化。采用秋水仙素阻抑实验鉴定单抗的染色体核型,Western blot和间接ELISA法鉴定单抗特异性、种属交叉反应、效价和亲和力,hAPE1 15肽阵列鉴定单抗的抗原表位。将单抗初步应用于免疫印迹、免疫细胞化学、免疫细胞荧光和免疫组织化学试验等实验中评价单抗性能。 3. hAPE1双抗夹心ELISA法在肿瘤血清学检测中的初步应用 对健康体检者、良性病变患者、肾癌、前列腺癌和睾丸癌患者的C-12多肿瘤标志物蛋白芯片检测结果进行回顾性分析,评价蛋白芯片的诊断作用;同时用hAPE1双抗夹心ELISA法对同批样本的血清标本进行检测,统计分析hAPE1的血清含量和诊断评价,并对两种检测结果进行比较,评价hAPE1在恶性肿瘤诊断中的意义。 研究结果 1. hAPE1原核表达载体的构建、融合蛋白的纯化及功能鉴定 所构建的pET28-hAPE1重组质粒经双酶切及DNA测序鉴定表明构建正确。pET28-hAPE1重组质粒在37℃,0.4mmol/L IPTG的诱导条件下可在E.coli BL21(DE3)中高效表达,蛋白含量可达菌体总蛋白量的50%,经SDS-PAGE及Western blot鉴定均在预期位置出现阳性条带;通过His亲和层析纯化得到纯度90%以上的hAPE1融合蛋白,经SDS-PAGE、Western blot及EMSA鉴定hAPE1融合蛋白具有抗原性、AP位点修复活性和氧化还原活性。 2. hAPE1单克隆抗体的制备、生物学特性鉴定及初步应用 经过4次细胞融合,成功获得了2株能稳定分泌hAPE1单克隆抗体的杂交瘤细胞株,命名为杂交瘤细胞2-G1和4-F6,并通过抗体纯化技术对大量制备的腹水进行浓缩和纯化,得到纯度90%以上的2-G1 mAb和4-F6 mAb。利用ELISA、Western blot等方法检测其效价超过1:107,Ig亚类分别是IgG2b和IgG3,亲和常数分别为1.8×10-7和3.4×10-5,能特异性识别hAPE1天然蛋白和融合蛋白,与兔、小鼠、大鼠无种属交叉反应。2-G1 mAb的抗原表位为构象型表位,可应用于免疫细胞化学、免疫细胞荧光和免疫组织化学等实验技术中。 3. hAPE1双抗夹心ELISA法在肿瘤血清学检测中的初步应用 健康体检者、良性病变患者、肾癌、前列腺癌和睾丸癌患者血清中hAPE1含量分别为8.97 (2.47,13.58) ng/ml、9.54 ( 2.36,14.22) ng/ml、14.98 (7.14,45.33) ng/ml、14.29 (10.83,33.68) ng/ml和20.74 (8.76,58.81) ng/ml,肿瘤患者与健康体检者比较差异有统计学意义( P 0.05 )。hAPE1双抗夹心ELISA法对肾癌、前列腺癌和睾丸癌的诊断阳性率分别为:57.14%、53.33%和66.67%,C-12多肿瘤标志物蛋白芯片对肾癌、前列腺癌和睾丸癌的诊断阳性率分别为:48.65%、80.00%和62.16%。结果表明,两种方法对3种肿瘤的诊断阳性率无统计学差异( P 0.05 )。除前列腺癌外,hAPE1与蛋白芯片联合检测肾癌和睾丸癌可显著提高诊断阳性率,具有统计学差异( P 0.05 )。 结论 1.成功构建hAPE1原核表达载体pET28a-hAPE1,可在E.coli BL21(DE3)中高效表达,经His亲和层析纯化得到hAPE1融合蛋白,纯度达90%以上,具有抗原性、AP位点修复活性及氧化还原活性。 2.成功获得2株稳定分泌抗hAPE1单克隆抗体的杂交瘤细胞株2-G1和4-F6,并纯化获得2株高纯度、高效价、高特异性和较高亲和力的hAPE1单克隆抗体2-G1 mAb和4-F6 mAb。其中2-G1 mAb的抗原表位为构象型表位,可用于多种免疫学实验中,是良好的检测试剂。 3.肾癌、前列腺癌和睾丸癌的血清hAPE1蛋白水平显著高于健康体检者, hAPE1双抗夹心ELISA法在恶性肿瘤诊断中具有临床意义,与C-12多肿瘤标志物蛋白芯片联合检测可提高对肾癌和睾丸癌的诊断阳性率。
[Abstract]:The early detection and diagnosis of tumor are of great importance to the prevention and treatment of tumor . As one of the conventional methods for tumor clinical diagnosis , tumor markers have been found to be of great significance to the early detection and prognosis of tumor .


Human apurinic / apyrimidinic endonuclease ( hAPE1 ) is a key enzyme in DNA damage repair ( BER ) pathway . It is a multi - functional protein which has a close relationship with cell apoptosis , proliferation , differentiation and transformation of tumor cells , sensitivity to radiotherapy and chemotherapy .


Purpose of study


1 . Construction of the prokaryotic expression vector of hAPE1 , induced expression , purification of hAPE1 fusion protein , and preliminary analysis of its biological function ;


2 . A hybridoma cell line stably secreting hAPE1 monoclonal antibody is established , the hAPE1 monoclonal antibody is prepared and purified , the biological characteristics thereof are identified and the preliminary application is carried out ;


3 . The diagnostic role of hAPE1 in the diagnosis of malignant tumor was investigated by using the hAPE1 serological test method , and the clinical significance of the combination of hAPE1 and multi - tumor marker protein chip in the diagnosis of malignant tumor was discussed .


Research content and methods


1 . Construction of Prokaryotic Expression Vector of hAPE1 , Purification and Functional Identification of Fusion Protein


The prokaryotic expression vector pET28a - hAPE1 containing the full - length gene sequence of hAPE1 was constructed by molecular cloning . After sequencing , the pET28a - hAPE1 recombinant plasmid was transformed into E . coli BL21 ( DE3 ) . The protein was purified by IPTG affinity chromatography . Western blot and EMSA were used to analyze the biological function of hAPE1 fusion protein .


2 . Preparation , Characterization and Preliminary Application of Monoclonal Antibodies to hAPE1


A hybridoma cell line secreting hAPE1 monoclonal antibody was obtained by using purified hAPE1 fusion protein as antigen in subcutaneous and intraperitoneal injection of Balb / c mice . The hybridoma cell line stably secreting hAPE1 monoclonal antibody was obtained by means of cell fusion , indirect ELISA and cloning . The monoclonal antibody was identified by Western blot and indirect ELISA . The monoclonal antibody was identified by Western blot and indirect ELISA .


Preliminary Application of 3 . hAPE1 Double Anti - Sandwich ELISA in the Detection of Tumor Serology


The detection results of C - 12 multi - tumor marker protein in patients with healthy physical examination , benign lesion , renal cancer , prostate cancer and testicular cancer were analyzed retrospectively , and the diagnostic function of protein chip was evaluated ; meanwhile , the serum samples of the same batch of samples were tested by using the hAPE1 double anti - sandwich ELISA method , and the serum levels and diagnostic evaluation of hAPE1 were statistically analyzed , and the significance of hAPE1 in the diagnosis of malignant tumor was evaluated .


Results of the study


1 . Construction of Prokaryotic Expression Vector of hAPE1 , Purification and Functional Identification of Fusion Protein


The recombinant plasmid pET28 - hAPE1 was identified by double digestion and DNA sequencing . The recombinant plasmid pET28 - hAPE1 was highly expressed in E . coli BL21 ( DE3 ) at 37 鈩，

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