Preparation Of Monoclonal Antibody Library Against Human FXYD6and Its Application On Diagnosis In Cholangiocarcinoma

Chapter I Prokaryotic expression and purification of FXYD6antigenObjective: 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 FXYD6Objective: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 cholangiocarcinomaObjective： 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.