人FXYD6单克隆抗体库的制备及其在胆管癌诊断中的应用
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摘要
第一部分人FXYD6抗原的表达与纯化
目的：构建含人FXYD6胞内区-G4S-FXYD6胞外区-G4S-GST-His6TAG基因序列的原核表达质粒，并诱导其表达，并对表达出的蛋白进行纯化。方法：构建去除FXYD6跨膜区域的FXYD6胞内区-G4S-FXYD6胞外区-G4S基因片段，插入原核表达载体pET30a-GST中，以重组表达质粒转化感受态大肠杆菌BL21，SDS-PAGE检测蛋白表达情况，重组蛋白经镍柱纯化后，SDS-PAGE检测蛋白纯度，BCA法检测浓度。结果：成功构建了原核表达载体pET30a-GST-FXYD6，诱导后重组蛋白大小与预期相符在33kD-45kD之间，其纯度在90%以上。结论：成功制备出了FXYD6重组蛋白，为制备FXYD6胞外区和胞内区的单克隆抗体库奠定了基础。
第二部分人FXYD6单克隆抗体库的制备与签定
目的：研制抗人FXYD6功能区单克隆抗体库，并对其进行签定及筛选分泌胞内、胞外区单克隆抗体的杂交瘤细胞株。方法：以去除跨膜区域FXYD6功能区重组蛋白作为免疫原免疫BALB/c小鼠，取其脾细胞与SP2/0骨髓瘤细胞融合，经多次筛选及克隆化，建立可稳定分泌抗人FXYD6胞外区及胞内区的单克隆抗体杂交瘤。用间接ELISA、蛋白免疫印迹签定特异性及亚型，再用间接ELISA法通过合成的胞外区多肽筛选出分泌胞外区、胞内区的FXYD6单克隆抗体的杂交瘤细胞株，用免疫组化方法筛选可识别天然构象的胞外区单克隆抗体，用腹水诱导法对胞外区单克隆抗体进行制备、纯化并检测亲和常数。用真核表达质粒pcDNATM3.1/myc-His(-)B-FXYD6瞬转上调FXYD6蛋白表达的293T细胞在体外检测胞外区抗体功能。结果：成功制备了6株能持续分泌的胞内区抗体的杂交瘤，2株持续分泌胞外区抗体的杂交瘤，这8株杂交瘤分泌的抗体可特异性识别天然构象的人FXYD6蛋白，2种胞外区单克隆抗体的亲和常数均在1010以上，这两种抗体均属于阻断功能性单抗。结论：成功制备了分泌抗人FXYD6胞外区及胞内区单克隆抗体的杂交瘤细胞库和制备了两种具有阻断性功能的胞外区单抗，为进一步研究其组织分布及作用机制签定了基础。
第三部分FXYD6在胆管癌中的表达及其生物学意义
目的：探讨FXYD6蛋白在胆管癌组织及相应远端正常胆管组织的表达情况，同时分析其在胆管癌中的表达与临床病理学特征的关系。方法：我们应用链酶亲和素-生物素-过氧化物酶复合物（SABC）免疫组织化学法检测72例胆管癌和远端正常胆管中FXYD6蛋白的表达，分析FXYD6表达与胆管癌临床病理学特征的关系。结果：FXYD6在胆管癌组织中的阳性表达率明显高于相应胆管组织的阳性表达率（69%vs.33.3%；p=0.002），而且FXYD6在高、中分化的胆管癌中的阳性表达明显高于低分化及粘液癌中的阳性表达（85.7%vs.40%；p=0.000）。FXYD6在正常胆管中的表达与解剖部位无关（p=0.945）。其在胆管癌中的表达与性别（p=0.393）、年龄（p=0.174）、组织学类型（p=0.123）、T分期（p=0.164）、淋巴结转移（p=0.343）、神经浸润（p=0.088）及肿瘤位置（p=0.238）无关。结论：FXYD6是胆管癌的一种新型肿瘤生物标记物，与胆管癌分化程度相关，其在胆管癌中的高表达可能提示较好的临床预后。
Chapter I Prokaryotic expression and purification of FXYD6antigen
Objective: To construct prokaryotic expression plasmids of FXYD6which dose not contain transmembrane region of the protein and to purify the proteinexpressed. Methods: The coding sequence of FXYD6which does not containtransmembrane region synthesized and cloned into the prokaryotic expression vectorpET-30a-GST. The6×His-tagged protein was induced by IPTG in E.coli BL-21and therecombinant protein was identified by SDS-PAGE. After the protein was purified by nickelcolumn, the purity of protein was tested by SDS-PAGE, the concentration was detected byBCA methods. Results: The FXYD6gene fragment was synthesized and thepET-30a-GST-FXYD6was successfully constructed. The recombinant protein FXYD6washighly expressed in E.coli with a molecule mass which conformed to the expected. Thepurity of the protein was more than90%. Conclusion: FXYD6antigen was successfullyexpressed by prokaryotic expression system and the proten with high purity was got.
Chapter II Preparation and identification of monoclonal antibodylibrary against human FXYD6
Objective:To prepare and identify the monoclonal antibody libraryagainst human FXYD6, and to screen the monoclonal antibodies against intracellular andextracellular region of human FXYD6. Methods: FXYD6recombinant protein was usedto immunize Balb/c mice, and splenocytes isolated from Balb/c mice were fused withmouse myeloma cells SP2/0. After several rounds of screening and cloning, established theantibodies against the extracellular domain and the intracelluar domain of human FXYD6.The FXYD6antibodies which only identifed the FXYD6recombinant protein, and the immunoglobulin subtype were screened with indirect ELISA. The specificity wasevaluated with Western blot. We employed extracellular region polypeptide todistinguished monoclonal antibodies against extracelluar and intracellular region withindirect ELISA. The antibodies which could identify the antigen with native conformationwas screened with immunohistochemistry. The antibodes againt extracelluar region wasprepared with ascites induced methods, and purificated with protein G sepharose affinitycolumn. And the affinity constants was examined with indirect ELISA. The293T cells, inwhich FXYD6were up-regulated with transient transfection by pcDNATM3.1/myc-His(-)B-FXYD6eukaryotic plasmid, were used to detect the function of the purificated mAb.Result: We obtained8cell lines of hybridoma, by6of which the antibodies secreted wereagainst intracelluar region and the rest were against extracelluar region. These8kinds ofhybridoma all secreted IgG antibodies, which could identify the FXYD6protein withnative conformation. And the affinity constants of antibodies which were againstextracelluar region were both1010above. The purified monoclonal antibodies wereinhibitory antibodes. Conclution: We had generated8cell lines of hybridoma by which theantibodies were secreted could identify the antigen with native conformation, and prepared2kinds of extracelluar and funtional antibodies, which provided the basis for further studyof the tissue distribution of FXYD6protein and its specific mechanism.
Chapter III FXYD6expression and biological significancein cholangiocarcinoma
Objective： To investigate the expression of FXYD6protein incholangiocarcinoma tissues and distal normal bile duct matched with the tumors and therelationship of FXYD6protein expression with the clinical and pathological characteristicsof cholangiocarcinoma. Methods: We exmined the expression of FXYD6protein in72cholangiocarcinoma and distal normal duct matched with the tumors with Streptavidin-biotin complex (SABC) immunohistochemistry. And analysed the relationship ofFXYD6protein expression with the biological characteristics of cholangiocarcinoma wasalso analysed. Results: The positive expression rate of FXYD6was statistically higher incholangiocarcinoma than that in normal bile duct (69%vs33.3%, p=0.002). Furthermore,the positive expression rate of FXYD6in well and moderately differentiated cholangiocarcinoma was obvious higher than that in poorly differentiated and mucinouscholangiocarcinoma (85.7%vs40%, p=0.000). Its expression in normal bile duct was notrelated with the anatomic position (p=0.945). And there was no significant correlationbetween the expression of FXYD6in cholangiocarcinoma and gender (p=0.393), age(p=0.174), histological type (p=0.123), T stage (p=0.164), lymph node metastasis(p=0.343), perineural invasion (p=0.088), and tumor location (p=0.238). Conclustion:FXYD6might be a new biomarker of cholangiocarcinoma and associated with a favorableprognosis in the malignant disease.
目的：构建含人FXYD6胞内区-G4S-FXYD6胞外区-G4S-GST-His6TAG基因序列的原核表达质粒，并诱导其表达，并对表达出的蛋白进行纯化。方法：构建去除FXYD6跨膜区域的FXYD6胞内区-G4S-FXYD6胞外区-G4S基因片段，插入原核表达载体pET30a-GST中，以重组表达质粒转化感受态大肠杆菌BL21，SDS-PAGE检测蛋白表达情况，重组蛋白经镍柱纯化后，SDS-PAGE检测蛋白纯度，BCA法检测浓度。结果：成功构建了原核表达载体pET30a-GST-FXYD6，诱导后重组蛋白大小与预期相符在33kD-45kD之间，其纯度在90%以上。结论：成功制备出了FXYD6重组蛋白，为制备FXYD6胞外区和胞内区的单克隆抗体库奠定了基础。
第二部分人FXYD6单克隆抗体库的制备与签定
目的：研制抗人FXYD6功能区单克隆抗体库，并对其进行签定及筛选分泌胞内、胞外区单克隆抗体的杂交瘤细胞株。方法：以去除跨膜区域FXYD6功能区重组蛋白作为免疫原免疫BALB/c小鼠，取其脾细胞与SP2/0骨髓瘤细胞融合，经多次筛选及克隆化，建立可稳定分泌抗人FXYD6胞外区及胞内区的单克隆抗体杂交瘤。用间接ELISA、蛋白免疫印迹签定特异性及亚型，再用间接ELISA法通过合成的胞外区多肽筛选出分泌胞外区、胞内区的FXYD6单克隆抗体的杂交瘤细胞株，用免疫组化方法筛选可识别天然构象的胞外区单克隆抗体，用腹水诱导法对胞外区单克隆抗体进行制备、纯化并检测亲和常数。用真核表达质粒pcDNATM3.1/myc-His(-)B-FXYD6瞬转上调FXYD6蛋白表达的293T细胞在体外检测胞外区抗体功能。结果：成功制备了6株能持续分泌的胞内区抗体的杂交瘤，2株持续分泌胞外区抗体的杂交瘤，这8株杂交瘤分泌的抗体可特异性识别天然构象的人FXYD6蛋白，2种胞外区单克隆抗体的亲和常数均在1010以上，这两种抗体均属于阻断功能性单抗。结论：成功制备了分泌抗人FXYD6胞外区及胞内区单克隆抗体的杂交瘤细胞库和制备了两种具有阻断性功能的胞外区单抗，为进一步研究其组织分布及作用机制签定了基础。
第三部分FXYD6在胆管癌中的表达及其生物学意义
目的：探讨FXYD6蛋白在胆管癌组织及相应远端正常胆管组织的表达情况，同时分析其在胆管癌中的表达与临床病理学特征的关系。方法：我们应用链酶亲和素-生物素-过氧化物酶复合物（SABC）免疫组织化学法检测72例胆管癌和远端正常胆管中FXYD6蛋白的表达，分析FXYD6表达与胆管癌临床病理学特征的关系。结果：FXYD6在胆管癌组织中的阳性表达率明显高于相应胆管组织的阳性表达率（69%vs.33.3%；p=0.002），而且FXYD6在高、中分化的胆管癌中的阳性表达明显高于低分化及粘液癌中的阳性表达（85.7%vs.40%；p=0.000）。FXYD6在正常胆管中的表达与解剖部位无关（p=0.945）。其在胆管癌中的表达与性别（p=0.393）、年龄（p=0.174）、组织学类型（p=0.123）、T分期（p=0.164）、淋巴结转移（p=0.343）、神经浸润（p=0.088）及肿瘤位置（p=0.238）无关。结论：FXYD6是胆管癌的一种新型肿瘤生物标记物，与胆管癌分化程度相关，其在胆管癌中的高表达可能提示较好的临床预后。
Chapter I Prokaryotic expression and purification of FXYD6antigen
Objective: To construct prokaryotic expression plasmids of FXYD6which dose not contain transmembrane region of the protein and to purify the proteinexpressed. Methods: The coding sequence of FXYD6which does not containtransmembrane region synthesized and cloned into the prokaryotic expression vectorpET-30a-GST. The6×His-tagged protein was induced by IPTG in E.coli BL-21and therecombinant protein was identified by SDS-PAGE. After the protein was purified by nickelcolumn, the purity of protein was tested by SDS-PAGE, the concentration was detected byBCA methods. Results: The FXYD6gene fragment was synthesized and thepET-30a-GST-FXYD6was successfully constructed. The recombinant protein FXYD6washighly expressed in E.coli with a molecule mass which conformed to the expected. Thepurity of the protein was more than90%. Conclusion: FXYD6antigen was successfullyexpressed by prokaryotic expression system and the proten with high purity was got.
Chapter II Preparation and identification of monoclonal antibodylibrary against human FXYD6
Objective:To prepare and identify the monoclonal antibody libraryagainst human FXYD6, and to screen the monoclonal antibodies against intracellular andextracellular region of human FXYD6. Methods: FXYD6recombinant protein was usedto immunize Balb/c mice, and splenocytes isolated from Balb/c mice were fused withmouse myeloma cells SP2/0. After several rounds of screening and cloning, established theantibodies against the extracellular domain and the intracelluar domain of human FXYD6.The FXYD6antibodies which only identifed the FXYD6recombinant protein, and the immunoglobulin subtype were screened with indirect ELISA. The specificity wasevaluated with Western blot. We employed extracellular region polypeptide todistinguished monoclonal antibodies against extracelluar and intracellular region withindirect ELISA. The antibodies which could identify the antigen with native conformationwas screened with immunohistochemistry. The antibodes againt extracelluar region wasprepared with ascites induced methods, and purificated with protein G sepharose affinitycolumn. And the affinity constants was examined with indirect ELISA. The293T cells, inwhich FXYD6were up-regulated with transient transfection by pcDNATM3.1/myc-His(-)B-FXYD6eukaryotic plasmid, were used to detect the function of the purificated mAb.Result: We obtained8cell lines of hybridoma, by6of which the antibodies secreted wereagainst intracelluar region and the rest were against extracelluar region. These8kinds ofhybridoma all secreted IgG antibodies, which could identify the FXYD6protein withnative conformation. And the affinity constants of antibodies which were againstextracelluar region were both1010above. The purified monoclonal antibodies wereinhibitory antibodes. Conclution: We had generated8cell lines of hybridoma by which theantibodies were secreted could identify the antigen with native conformation, and prepared2kinds of extracelluar and funtional antibodies, which provided the basis for further studyof the tissue distribution of FXYD6protein and its specific mechanism.
Chapter III FXYD6expression and biological significancein cholangiocarcinoma
Objective： To investigate the expression of FXYD6protein incholangiocarcinoma tissues and distal normal bile duct matched with the tumors and therelationship of FXYD6protein expression with the clinical and pathological characteristicsof cholangiocarcinoma. Methods: We exmined the expression of FXYD6protein in72cholangiocarcinoma and distal normal duct matched with the tumors with Streptavidin-biotin complex (SABC) immunohistochemistry. And analysed the relationship ofFXYD6protein expression with the biological characteristics of cholangiocarcinoma wasalso analysed. Results: The positive expression rate of FXYD6was statistically higher incholangiocarcinoma than that in normal bile duct (69%vs33.3%, p=0.002). Furthermore,the positive expression rate of FXYD6in well and moderately differentiated cholangiocarcinoma was obvious higher than that in poorly differentiated and mucinouscholangiocarcinoma (85.7%vs40%, p=0.000). Its expression in normal bile duct was notrelated with the anatomic position (p=0.945). And there was no significant correlationbetween the expression of FXYD6in cholangiocarcinoma and gender (p=0.393), age(p=0.174), histological type (p=0.123), T stage (p=0.164), lymph node metastasis(p=0.343), perineural invasion (p=0.088), and tumor location (p=0.238). Conclustion:FXYD6might be a new biomarker of cholangiocarcinoma and associated with a favorableprognosis in the malignant disease.
引文
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