抗SARS-CoV抗原的人源Fab段噬菌体抗体库的构建

目的 :利用抗SARS冠状病毒IgG抗体阳性的SARS康复患者外周血淋巴细胞 ,构建人源Fab段抗体文库。方法 :制备外周血淋巴细胞总RNA ,逆转录成cDNA。以其为模板 ,利用针对家族特异性Ig基因的引物扩增重链Fd段和轻链基因 ,并重组到噬菌粒载体pComb3中 ,将重组噬菌粒载体电转化大肠杆菌XL 1Blue,酶切鉴定抗体库的重组率 ,并测定噬菌体抗体库的库容量。结果 :构建了源于SARS康复患者血清中抗Fab段的抗体文库 ,轻链、重链Fd段基因的重组率分别为91%和 75 % ,库容量为 7.2 3× 10 7。结论 :成功地构建了抗SARS CoV抗原的人源Fab段噬菌体抗体库

Construction of phage antibody library for Fab fragment from a convalescent patient infected with SARS coronavirus

AIM:To construct the phage antibody library for Fab fragment from a convalescent patient infected with SARS coronavirus. METHODS: Total RNA was extracted from peripheral blood lymphocytes of a patient with SARS virus-specific IgG antibody and was transcribed reversely into cDNA. Using the cDNA as template, Fd and light chain of Fab genes were amplified by PCR with specific primer and were cloned into phagemid pComb3. Then the recombinant phagemid was electroporated into E.coli XL-1 Blue. The recombinant rate was detected by enzyme digestion analysis, and library repertoire was determined. RESULTS: The antibody library of Fab fragment from a convalescent patient infected with SARS conronavirus was constructed. The recombinant rate of light chain and heavy chain of Fd gene were 91% and 75%, respectively. The library size was 7.23 x 10(7). CONCLUSION: Phage antibody library for human Fab fragment has been constructed successfully, which lays the foundation for further study.