中文摘要：

构建大容量核糖体展示抗克伦特罗单链抗体（single-chain fragment variant,scFv）文库,为进一步筛选抗体奠定基础。用克伦特罗人工抗原免疫小鼠,分离其脾细胞;Trizol法抽提总RNA,反转录成cDNA,PCR及重叠延伸PCR法构建核糖体展示scFv文库。再通过PCR和重叠延伸PCR在scFv文库序列5＇端添加T7启动子、茎环结构、核糖体结合位点,3＇端添加间隔区序列（T20-V109）、茎环结构构建核糖体展示抗克伦特罗scFv文库。核糖体展示scFv文库与Easy T3载体连接,转化大肠杆菌DH5α感受态细胞,经蓝白斑筛选,挑取5个阳性克隆双酶切和测序鉴定。结果表明：用于建库材料的免疫小鼠效价为1：128000;成功扩增重链可变区基因大约为360bp和轻链可变区基因大约为330pb;核糖体展示scFv文库基因大约1200bp;库容量达1.75×1013;阳性重组质粒均含有1200bp插入片段;利用IMGT/V-QUEST数据库对单链抗体可变区进行序列分析结果表明,重链可变区和轻链可变区基因均由胚系基因重排得到,且与鼠源胚系基因同源率都达84%以上;氨基酸序列比对结果表明,重链可变区氨基酸序列同源率为37.82%,轻链可变区氨基酸序列同源率为39.09%。其中多样性主要发生在VH-CDR3、VL-CDR1、VL-CDR2和VL-CDR3。成功构建了大容量抗克伦特罗单链抗体核糖体展示文库。

英文摘要：

In order to construct a high-capacity ribosome display anti-clenbuterol single chain Fv（CBLscFv） library for the selection of high affinity scFv antibody,CBL-BSA immunogen was immunized to Balb/c mice.Spleen cells were isolated and total RNA was extracted by the Trizol method for cloning heavy chain and light chain gene by RT-PCR.VH and VL were rearranged randomly by SOEing（splicing by overlap extension）.Finally,the elements T7 promoter,3＇ stem loop structure,ribosome binding sites,spacers and 5＇ stem loop structure were screened in vitro.The constructed library was verified by blue/white screening and further sequencing.The results showed that the titer of antiserum from immunized mice for library construction was 1：128000.The heavy and light chain variable region genes were successfully amplified to be approximately 360 bp and 330 bp,respectively.The fragment of the constructed ribosome display library was approximately 1200 bp with a capacity of 1.75 × 1013.Meanwhile,sequence analysis was carried out using IMGT/V-QUEST database.The results showed that the heavy chain and light chain variable region genes were rearranged by mouse IG set,and the homology rates were more than 84%.Amino acid sequence alignment revealed a homology rate of 37.82% for heavy chain amino acid sequences and 39.09% for light chain amino acid sequences.The diversity was observed in VH-CDR3,VL-CDR1,VL-CDR2 and VL-CDR3.Therefore,a high-capacity ribosome display library with the function for antibody screening was successfully constructed.