中文摘要：

目的构建天然人源甲状腺未分化癌噬菌体单链抗体库并初步筛选出抗甲状腺未分化癌抗体。方法从甲状腺未分化癌患者癌周淋巴结组织中提取总RNA,采用RT-PCR技术扩增抗体可变区基因VH和VL基因片段,并分别与相应Linker连接,再通过重叠延伸PCR技术将其拼接组装成sc Fv基因片段,并引入酶切位点SfiⅠ和NotⅠ。将双酶切后的sc Fv重组基因片段与噬菌粒载体p CANTAB-5E连接,产物经化学转化转入大肠杆菌TG1,超感染噬菌体,构建噬菌体单链抗体库。以甲状腺未分化癌细胞（ARO细胞株）为抗原对该抗体库进行4轮＂吸附-洗脱-扩增＂的筛选,并行ELISA鉴定抗体特异性。结果成功获得约为750 bp的sc Fv基因。用p UC19标准质粒测定转化效率达到108 cfu/μg,双酶切鉴定sc Fv基因的阳性插入率为86.4%（19/22）。筛选过程中,甲状腺未分化癌单链抗体得到富集,收获率不断提高,第4轮为第1轮的77倍。ELISA结果显示筛选出的抗体能与甲状腺ARO细胞株特异性结合。结论成功构建了人源甲状腺未分化癌噬菌体单链抗体库,并从中筛选出具有甲状腺未分化癌细胞特异性的人源噬菌体单链抗体,为进一步放免研究打下基础。

英文摘要：

The study designed to identify and construct a human natural phage single-chain antibody（sc Fv）library of human anaplastic thyroid carcinoma（ATC） using phage display technology. Total RNA was extracted from lymphatic tissue near ATC and used to amplify fragments consisting of a variable heavy chain（VH） and a variable light chain（VL） with reverse transcription polymerase chain reaction（PCR） and join with their linkers. After purification, VHand VLwere used to produce the sc Fv fragments using splicing-overlap-extension（SOE） PCR, and then loaded with the restricted enzyme cutting sites SfiⅠ and NotⅠ. The sc Fv gene was cloned in the p CANTAB-5E plasmid, and the recombinant phagemids were transformed to susceptible Escherichia coli TG1. Panning against ARO cell line was performed for four rounds and the antibody library was identified by ELSIA. After infection by the helper phage M13K07, the human ATC phage antibody library was successfully constructed, and the sc Fv gene was approximately 750 bp. The conversion efficiency measured by p UC19 standard plasmid was 108CFU/μg, and the positive insert ratio was 86.4%（19/22）. Moreover, the antibody was enriched with the increase of panning rounds, and the fourth one harvest yielded 77 times as much as that of first one. The ELISA results showed that the soluble sc Fv was highly active and could bind ARO cells, but not TT cells. In conclusion, a human ATC sc Fv antibodies gene library has successfully constructed and the selected sc Fv gene can specifically bind to human ARO cell line, which would provide an experimental basis for further radioimmunoimaging of the phage single-chain antibody with cell-specific ATC.