BSA-Y-DOTA免疫噬菌体Fab抗体库的构建及鉴定

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

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

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 sequenceing 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 κ 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 8×10 7, the recombination 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.