CAJ | 학술논문

目的构建人Tamm-Horfall蛋白（ THP）抗原决定簇的原核表达载体，表达纯化重组人Tamm-Horfall片段蛋白并制备单克隆抗体。方法将人 Tamm-Horfall 蛋白片段 cDNA 克隆至原核表达载体pET28a，经大肠杆菌表达、纯化，获得的Tamm-Horfall蛋白片段免疫小鼠，取其脾淋巴细胞与sp2/0细胞融合，制备能产生THP蛋白单克隆抗体的杂交瘤细胞株，进一步采用Western blot、ELISA、免疫组化等技术对制备的单克隆抗体进行鉴定。结果原核表达重组质粒在大肠杆菌中能高效表达THP蛋白抗原决定簌的蛋白片段，用纯化的THP蛋白片段制备了鼠抗THP单克隆抗体。结论成功制备了9株THP单克隆抗体，为进一步研究THP蛋白的分布、结构、功能及检测试剂盒的研发奠定了基础。
목적구건인Tamm-Horfall단백（ THP）항원결정족적원핵표체재체，표체순화중조인Tamm-Horfall편단단백병제비단극륭항체。방법장인 Tamm-Horfall 단백편단 cDNA 극륭지원핵표체재체pET28a，경대장간균표체、순화，획득적Tamm-Horfall단백편단면역소서，취기비림파세포여sp2/0세포융합，제비능산생THP단백단극륭항체적잡교류세포주，진일보채용Western blot、ELISA、면역조화등기술대제비적단극륭항체진행감정。결과원핵표체중조질립재대장간균중능고효표체THP단백항원결정속적단백편단，용순화적THP단백편단제비료서항THP단극륭항체。결론성공제비료9주THP단극륭항체，위진일보연구THP단백적분포、결구、공능급검측시제합적연발전정료기출。
Objective To construct expression vectors of human Tamm-Horfall protein antigenic determinant , to purify recombinant human Tamm-Horfall protein and to prepare monoclonal antibodies . Methods cDNA of human Tamm-Horfall protein fragment was cloned into pET 28 a expression vector and was further transfected into DE3.The recombinant protein fragment of human Tamm-Horsfall was used immuned Balb/c mouse. The spleen lymphocyte was fused with sp 2/0 hybridoma to produce monoclonal antibody against THP protein .Western blot,ELISA and immunohistochemistry were carried out to identify the monoclonal antibody produced by these hybridomas.Results Protein fragment of THP protein antigenic determinant was purified from DE 3 transfected with THP protein antigenic determinant cDNA prokaryotic expression vector ,and purified THP protein fragment was used to prepare THP monoclonal antibody against rats .Conclusions We successfully prepare nine hybridomas which can stably produce monoclonal antibodies against THP ,and these work has laid foundation for further study regarding THP proteins distribution ,structure,function and for detection kits .