【摘要】 目的构建库容量大、多样性好的核糖体展示单链抗体库,为进一步筛选单链抗体奠定基础。方法对2006年10月1日至11月31日收集于中山大学附属第二医院的人外周血(健康成人2名,胃癌3例,肠癌3例,胰腺癌1例,每例各5mL,新生儿2名各2mL)分离淋巴细胞,提取RNA;利用RT-PCR克隆出全套重链可变区基因(variable region of heavy chain,VH)、轻链可变区基因(variable region of light chain,VL);然后利用重叠延伸PCR技术连接构建VH-VL单链抗体库。并通过连接T-Vector转化E.coli JM109大肠埃希菌,经蓝白筛选,挑取阳性克隆测序以鉴定单链抗体组装。结果试验成功构建了单链抗体核糖体展示模板,其库容达1.1×1013。结论大容量核糖体展示单链抗体库的构建为筛选多种人源性单链抗体奠定了基础。更多还原

【Abstract】 Objective To construct high-capacity ribosome display single-chain Fv library for selection of high affinity ScFv antibody.Methods We isolate human lymphocyte from peripheralblood(2 normal,3 gastric cancer,3 colonic cancer,1 pancreatic cancer,each 5 mL and 2 newborn,each 2 mL)and extract RNA for cloning whole human heavy chain and light chain gene by RT-PCR.VH and VL were rearranged randomly by SOEing(splicing by overlap extension,SOEing).Finally,the elements for in vitro screening such as T7 promoter and ribosome binding site were introduced while the SOEing products were amplified.Moreover,ribosome display template were verified by blue/white screening and further sequencing.Results We successfully constructed ribosome display ScFv library with a volume of 1.1×1013.Conclusion The construction of high-capacity ScFv library shed light on multiple therapeutic ScFv screening.更多还原