【摘要】 目的　从构建的A型流感病毒核蛋白(Nucleoprotein)噬菌体单链抗体库中筛选阳性克隆,在大肠杆菌HB2 15 1中表达流感病毒核蛋白噬菌体单链抗体,为研究A型流感病毒免疫渗漏试剂盒建立基础。方法　经过连续三轮固相“亲和-洗脱-富集”筛选,丰富噬菌体抗体库中阳性克隆的比例,采用ELISA、SDS PAGE和Western blot方法对大肠杆菌培养上清液和细菌周质腔蛋白进行分析目的产物。采用亲和层析方法对细菌周质腔蛋白进行分离纯化并进行单链抗体滴度分析。结果　从96个克隆中筛选到10个阳性克隆,其中三个阳性信号较强,分别为A1、E1和G3。其A4 50 分别达到0 . 4 6 9、0 . 5 82和0 . 5 0 7。A型流感病毒核蛋白噬菌体单链抗体主要表达于细菌周质腔中,亲和纯化产物稀释4 0 .96倍,ELISA结果仍为阳性。结论　利用噬菌体抗体库技术,初步在大肠杆菌中表达了A型流感病毒噬菌体单链抗体。更多还原

【Abstract】 Objective To expression the phage single chain antibody against nucleoprotein of influenza virus A type in E.coli strain HB2151 by screening the positive clone from the phage antibody library against nucleoprotein, which will prepare for the construction of fast leak kit of Influenza virus A type. Methods The positive clone ratio in the phage antibody library was enriched by three-round continual solid panning, ELISA、SDS-PAGE and Western-blot methods were used to analyze the protein secreted into the supernants and periplasmic. Affinity chromatography was used to purified the protein expressed in periplasmic and the titer was also analyzed using ELISA method. Results 10 clones were screened from 96 clones and among the 10, there were 3 stronger positive with OD450nm value of 0.469、0.582 and 0.507, respectively. The phage single chain antibody against Influenza virus A type was primary expressed in periplasmic. The ELISA results were positive even if the single chain antibody purified by affinity chromatography was diluted 4096 folds. Conclusion Using phage antibody library technique, phage single antibody against nucleoprotein of influenza virus A type was preliminary expressed in E.coli.更多还原