中文摘要：

目的构建大容量（〉10^8）的人源天然噬菌体抗体库。方法提取人外周血淋巴细胞mRNA，反转录出cDNA第一链，以此为模板，PCR扩增重链（VH）和轻链（VL）基因片段。将VL基因PCR产物插入含Loxp和Loxp511序列的pDF载体中，电转化大肠杆菌XL1-Blue，构建轻链库，再将VH基因PCR产物插入轻链库载体中，电转化大肠杆菌XL1-Blue，构建初级库。进一步用初级库超感染BS1365菌，使其VH与VL发生重组，获得重组库，再利用重组库感染大肠杆菌XL1-Blue，构建工作库。结果所有VL和VL亚类基因的扩增产物片段大小均与理论值相符，轻重链基因的克隆效率均为100％。初级库容量为10^8，滴度为6×10^13；重组后的工作库有效容量为8×10^10，滴度至少为1×10^13。结论已构建容量为10m的人源天然噬菌体抗体库。

英文摘要：

Objective To establish a large capacity human naive phage antibody library. Methods The mRNA was extracted from human peripheral blood lymphocytes and transcribed to the first chain of cDNA, which was further used as a template for amplification of VH and VL gene fragments by PCR. Insert the PCR product of VL gene into the pDF vector containing Loxp and LoxpS11 sequences, and transform the constructed recombinant plasmid to E. coli XL1-Blue to establish light chain library. Insert the PCR product of VH gene into the vector for light chain library, and transform the constructed recombinant plasmid to E.coli XL1-Blue to establish primary phage antibody library. Super-infect E. coli BS1365 with the primary phage antibody library to establish recombinant phage antibody library. Infect E. coli XL1-Blue with the recombinant phage antibody library to establish working phage antibody library. Results All the lengths of amplified VH and VL subclass genes were consistent with the theoretical values. Both the cloning efficacies of VH and VL genes were 100%. The capacity and titer of primary phage antibody library were 8×10^8 and 6×10^13, and those of working phage antibody library were 8×10^10 and not less than 1×10^13 respectively. Conclusion A human naive phage antibody library with a capacity of 10^10 was established.