人源甲状腺髓样癌噬菌体抗体库的构建及初步鉴定

Construction of human medullary thyroid carcinoma phage antibody library and preliminary identification

摘要:

目的：应用噬菌体抗体展示技术，构建大容量天然人源甲状腺髓样癌（medullary thyroid carcinoma，MTC）噬菌体单链抗体库。方法：提取ＭＴＣ病人癌周淋巴结总RNA，通过RT-PCR方法获得抗体可变区基因VH和VL基因片断，以它们为模板分别扩增VH-Linker和VL-Linker，再用剪切重叠延伸ＰＣＲ技术将之拼接组装成单链抗体可变区基因片段（single chain fragment variable，scFv）后引入酶切位点SfiⅠ和NotⅠ。将scFv基因克隆入噬菌粒栽体pCANTAB-5E后经电转入大肠杆菌Ecoli TG1，之后经辅助噬菌体M13K07超感染，构建人源MTC噬菌体单链抗体库。PCR鉴定scFv片段阳性插入率，1.0%琼脂糖凝胶电泳鉴定阳性克隆双酶切产物。结果：MTC周围淋巴结总RNA的琼脂糖电泳结果可见清晰的28 S、18 S条带；VH基因的大小约为370 bp，VL基因为350 bp，组装后的scFv基因约为750 bp。用pUC19标准质粒测定转化效率达到108 cfu/μg，scFv的阳性插入率为87.5％（21/24）。结论：成功地构建了人源MTC噬菌体展示文库，为进一步筛选具有MTC细胞特异性的人源噬菌体单链抗体奠定了实验基础。

Abstract:

Objective：To construct natural human medullary thyroid carcinoma phage antibody library using phage display technology. Methods：Total RNA was extracted from the lymphatic tissue around medullary thyroid carcinoma. VH and VL fragments were amplified with RT-PCR，which were used as templates to amplify VH-Linker and VL-Linker. Then restricted enzyme cutting sites sfi Ⅰ and NotⅠ after single chain fragment variable（scFv） gene fragments was assembled with splicing-overlap-extension PCR. The scFv gene was cloned in pCANTAB-5E plasmid then transferred to Ecoli TG1. After the helper phage M13K07 infection，a human phage antibody li－brary was constructed. The insertion ratio of scFv gene was identified with PCR and the double enzyme digestion products of positive clones were analyzed with 1.0% agarose gel electrophoresis. Results：Clear 28 S and 18 S strips were found in total RNA；size of VH gene was about 370 bp，of VL gene was about 350 bp and of scFv gene was about 750 bp. Determination of conversion efficiency reached 108 cfu/μg with pUC19 standard plasmid and the positive insert ratio was 87.5%（21/24）. Conclusions：Human medullary thyroid carcinoma phage display library is constructed successfully. It could provide experimental basis for further screening of people with medullary thyroid cancer cell-specific phage single-chain antibody.