人源性老年痴呆单链噬菌体抗体库的构建

The construction of human phage display library of Alzheimer's Disease

目的构建人源性老年痴呆(AD)单链噬菌体抗体库,为进一步筛选AD相关抗原的人源性抗体奠定基础。方法从200 ml AD病人的外周血中分离B淋巴细胞,提取总RNA,经逆转录合成总cDNA。以PCR技术,利用特定的引物分别扩增出人抗体的重链和轻链可变区基因片段(VH、VL),并分别克隆入噬菌粒pDAN5中,构建出含人单链抗体可变区(scFv)基因序列的pDAN5克隆载体,电转化感受态大肠杆菌XLI-Blue后,经辅助噬菌体M13KO7超感染后回收全部重组噬菌体,构建成初级噬菌体抗体库。从抗体库中随机挑选数个克隆,提取质粒,PCR扩增目的片段后,用内切酶BstNⅠ消化,每个克隆经消化后的DNA指纹印迹用琼脂糖凝胶电泳分析,评价抗体库的多样性。结果所有抗体VH和VL基因片段均得到了扩增并成功克隆及转化,经辅助噬菌体感染后,构建成初级抗体库。BstNⅠ消化后的DNA指纹图谱显示各克隆抗体基因各不相同,经计算构建的噬菌体抗体库容量为1.5×106。结论利用噬菌体抗体库技术成功构建了AD病人的单链可变区噬菌体抗体库。

Objective To construct an human phage-display library of Alzheimer＇s disease in order to provide the basis of screening specific antibodies against the relevant antigens of AD.Methods The B lymphocytes were isolated from 200 ml peripheral blood of AD patients,and total RNA of B cells was prepared followed by cDNA synthesis by reverse transcription.The VH（heavy chain variable region） and VL（light chain variable region） genes were amplified from cDNA with polymerase chain reaction（PCR） using specific primers,and were cloned into the phagemid,pDAN5,respectively.The recombinant pDAN5 containing the genes of human single-chain variable fragments was constructed and then transformed into Escherichia coli XL1-Blue by electroporation.All the positive colonies were collected by scraping,superinfected by helper phage M13KO7,and reclaimed the recombinant phage,finally stored as a primary scFv library.A number of individual clones from the primary library were randomly picked and their phagemids with scFv were prepared.After amplifying with PCR,the individual scFv gene was digested by BstNI,and the fingerprinting of each colony was analyzed by agarose gels in order to evaluate the diversity of the library.Results All the VH and VL gene segments were amplified,successfully cloned into the vectors and transformed into the E.coli.After infected by helper phage M13KO7,the primary scFv library was constructed.Each random selected clone from the library showed a unique BstNI–digested fingerprinting pattern,indicating that individual clones in the library were different.This library was calculated to have a diversity of 1.5×10^6 members.Conclusions A human phage-display scFv antibody library of AD is constructed successfully by using phage-display techniques.