米尔比霉素肟化物ScFv噬菌体展示抗体库的构建

Construction of ScFv phage display antibody library against milbemycin oxime

目的构建具16元大环内酯共性结构小分子物质的特异性抗体库。方法免疫原MILO-BSA免疫小鼠后从脾细胞中提取总RNA,RT-PCR扩增得到全套抗体重链可变区基因(VH)及轻链可变区基因(VL),SOE-PCR将VH、VL片段拼接扩增得到单链抗体可变区基因片段(ScFv)。将ScFv基因克隆到噬菌粒载体pCANTAB5E中,电转化感受态大肠杆菌,辅助噬菌体超感染得到上清液即为噬菌体抗体库。结果RT-PCR扩增出长360bp左右的抗体重链可变区基因(VH)及340bp左右的轻链可变区基因(VL),SOE-PCR扩增得到750bp左右的ScFv基因片段,成功构建了库容量为2.4×106,滴度为2.0×1012pfu/mL的噬菌体单链抗体库。结论构建的抗体库目的片段连接率较高,多样性较好,为下一步16元大环内酯类小分子物质高特异性抗体的筛选奠了基础。

Objective To construct a specific phage display library for panning antibodies against compounds of 16-membered macrocyclic backbone. Methods The total RNA was extracted from spleen cells of mouse immunized with milbemycin oxime （MILO）-BSA . RT-PCR was used to amplify immunoglobulin variable heavy chain region genes （VH） and variable hght chain region genes （VL）. The purified VH and VL genes were jointed together through splicing by overlap extension （SOE）, using the fragment-containing parts of the linker. The ScFv genes were generated by PCR, using the primers contained the slice sites of restriction endonuclease. The ScFv gene fragments have been ligated into the phage vector pCANTABSE and were transformed into competent E. coli TG1 cells. The transformed bacterium was infected with helper phage, and then the cell was sedimented and the supematant was collected as the library. Results VH, VL, and ScFv genes were about 360, 340, and 750 bp amplified by RT-PCR and SOE-PCR. A phage display antibody library contain 2.4×10^6 clones and 2.0×10^12 pfu/mL bacteriophage plaques has been successfully constructed. Conclusion The ligation efficiency and the diversity of the library are satisfying. The research is helpful for establishing new panning methods of antibody library and getting high affinity antibody against small molecules including 16-membered macrocyclic backbone.