BSA-Y-DOTA免疫噬菌体Fab抗体库的构建及鉴定

目的 :构建钇 十二烷四乙酸 (Y DOTA)免疫噬菌体Fab抗体库。方法 :将牛血清白蛋白 (BSA)与DOTA交联 ,并与金属Y鳌合制备成BSA Y DOTA ,用其免疫BALB/c小鼠。检测抗血清的滴度后 ,分离抗体阳性的小鼠脾淋巴细胞 ,提取总RNA。利用RT PCR扩增全套重链Fd和轻链基因 ,依次插入经改造的噬菌体载体pComb3M的相应酶切位点 ,构建成Fab噬菌体抗体库。用酶切、序列测定及ELISA等方法 ,对重组率、多样性及Fab的展示情况进行鉴定。结果 :成功得制备了BSA Y DOTA交联物 ,并获得较好的免疫效果。免疫小鼠的全套重链Fd片段和轻链均得到正确扩增 ;Fd片断和轻链基因均插入到载体pComb3M中 ;Fab抗体库的库容量达 8× 10 7;重组率约为90 % ,且具有良好的抗体基因多样性。另外 ,Fab片段也被展示于噬菌体表面。结论 :成功地构建了半抗原Y DOTA的Fab噬菌体抗体库 ,为筛选Y DOTA特异性抗体奠定了基础

Construction and characterization of anti-DOTA-Y Fab immune phage antibody library

AIM: To construct a anti-dodecane-tertraacetic acid-yttrium(DOTA-Y) immune Fab phage antibody library. METHODS:BALB/c were immunized with BSA-Y-DOTA which was prepared by DOTA-conjugated BSA and chelated with Y. After determination of anti-serum, total RNA was extracted from splenic lymphocytes of immuned mice. The heavy chain Fd and light chain Kappa genes repertoires of immunoglobulin were amplified respectively by RT-PCR, and then the amplified products were cloned into the reconstructive phage vector pComb3M to construct anti-DOTA-Y Fab antibody. And then, the recombination rate, diversity and display of Fab antibody library were identified by restriction endonuclease digestion, DNA sequencing and ELISA. RESULTS: BSA-Y-DOTA was prepared successfully, and a higher titer of immune sera was achieved. The amplified gene fragments of Fd and Kappa chain by RT-PCR were correct and the length was with about 650 bp, and were inserted exactly. The sink size of Fab phage display library reached 8 x 10(7), the re-combination rate was about 90%, and it possesed great diversity. In addition, ELISA detection showed that there was Fab expression on the phage library. CONCLUSION: An immune Fab phage antibody library of DOTA-Y has been constructed successfully, which lays a solid foundation for screening specific anti-DOTA-Y antibody.