【摘要】 本研究旨在通过构建羊驼非免疫重链单域抗体库,完成抗体库多样性的鉴定,为进一步筛选抗原特异性重链抗体奠定基础。我们从未经免疫的羊驼外周血中分离外周血单个核细胞(PBMC),抽提RNA后,用RT-PCR方法特异性扩增羊驼重链抗体可变区(VHH)片段;并采用两步连接方法将重链抗体可变区片段与噬菌粒载体pCANTAB5E连接获得重组子,多次电转感受态大肠杆菌TG1后获得VHH抗体基因库;并采用稀释计数法测定抗体库库容量,随机挑取克隆测序验证抗体库多样性。结果显示,我们所构建的羊驼非免疫重链单域抗体库的库容量为1.5×109,随机克隆测序验证多样性良好,独立克隆所占比例为80%,并显示出和人源抗体较高的同源性。上述结果表明,我们已经成功构建获得大容量的羊驼非免疫重链单域抗体库,为进一步筛选抗原特异性重链抗体奠定基础。更多还原

【Abstract】 To construct a non-immune Llama variable domain of heavy chain antibody(VHH) phage display antibody library(VHH antibody library),Llama peripheral blood mononuclear lymphocytes were isolated from whole blood by Ficoll-hypaque density gradient centrifugation.Total RNA was extracted from PBMCs and reverse-transcripted into cDNA by using specific primers.VHH were amplified by nested PCR.PCR products of VHH fragments were then purified and ligated with phagemid vector pCANTAB5E by a modified two-step ligation method.Recombinant pCANTAB5E-VHH vectors were electroporated into competent TG1 E.coli cells to obtain the primary VHH antibody library.The library capacity was titrated through limited dilution.Recombination efficacy and diversity of VHH antibody library was determined by sequencing analysis.Alignment analysis was performed to compare the homology between Llama VHH domain and human/mouse variable regions of heavy chains.By using a modified strategy,we have constructed a non-immune Llama VHH antibody library with 1.5×109 capacity.The VHH sequences of randomly-selected 20 clones from the library indicated that the frequency of the correct and potentially functional clones was above 80%.VHH domains from Llama heavy chain antibody displayed high homology with that from human heavy chains.Taken together,a non-immune Llama VHH phage display antibody library was successfully constructed with large capacity and good diversity,which provides a useful platform to prepare VHH antibody interested for future application.更多还原