论文部分内容阅读
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
OBJECTIVE: To construct Y DOTA immunopharmaceutical Fab phage antibody library. Methods: Bovine serum albumin (BSA) was cross-linked with DOTA and chelated with metal Y to prepare BSA Y DOTA, which was used to immunize BALB / c mice. After detecting the antiserum titer, antibody-positive mouse spleen lymphocytes were isolated and total RNA was extracted. The complete set of heavy chain Fd and light chain genes were amplified by RT PCR and inserted into the corresponding restriction sites of the modified phage vector pComb3M to construct Fab phage antibody library. Restriction analysis, sequencing and ELISA were used to identify the recombination rate, diversity and Fab display status. Results: The cross-linked BSA Y DOTA was successfully prepared and the better immune effect was obtained. The complete heavy chain Fd fragment and the light chain of the immunized mice were correctly amplified; the Fd fragment and the light chain gene were both inserted into the vector pComb3M; the library capacity of the Fab antibody library was 8 × 10 7; the recombination rate was about 90% And has a good antibody gene diversity. In addition, Fab fragments are also displayed on the phage surface. Conclusion: The Fab phage antibody library of hapten Y DOTA was successfully constructed, which laid the foundation for the screening of Y DOTA-specific antibodies