抗大别班达病毒人源单克隆抗体Fab片段的噬菌体展示文库构建

Construction of phage display antibody library of human monoclonal Fab antibody against Dabie Bandavirus

目的构建具有潜在中和活性的人源抗大别班达病毒(SFTSV)抗体Fab片段的噬菌体展示文库。方法从具有交叉中和活性的SFTS恢复期患者血液样本中分离外周血单个核细胞(PBMC)并提取总RNA,逆转录PCR得到cDNA。以cDNA为模板进行第一轮PCR扩增,分别获得抗体重链可变区(VH)和轻链可变区(Vκ和Vλ)序列。以pComb3XTT、pComb3Xλ质粒为模板,分别获得抗体重链恒定区(CH1)和轻链恒定区(Cκ、Cλ)序列。第二轮通过重叠延伸PCR(SOE-PCR)扩增VH和CH1得到Fd重链,同样扩增Vκ和Cκ、Vλ和Cλ得到完整的κ和λ抗体轻链。第三轮以上述扩增产物为模板再次进行SOE-PCR,得到完整的抗体Fab基因片段。将Fab基因片段插入到pComb3XSS噬菌粒中,构建噬菌体展示抗体文库。结果通过三轮PCR扩增出抗体Fab片段的序列,成功构建库容量为1.875×10^(9)的噬菌体展示抗体文库。结论利用具有交叉中和活性的SFTS恢复期患者血源建立了人源抗SFTSV Fab片段的噬菌体展示抗体文库,为下一步筛选特异性单克隆中和抗体建立了可靠的前期基础。

Objective To construct a phage display antibody library of human anti-Dabie bandavirus(SFTSV)Fab fragment with potentially neutralizing activity.Methods Peripheral blood specimens of patients recovering from SFTS were collected,and peripheral blood mononuclear cells(PBMC)were isolated from the specimens with cross-neutralization activity.Then PBMCs were subjected to extract total RNA,and cDNA was obtained by reverse transcription PCR(RT-PCR)using these total RNAs as template.The first round PCR amplification of sequences of heavy chain variable region(VH)and light chain variable region(Vκ and Vλ)was finished using cDNA as template,similarly,heavy chain constant(CH_(1))and light chain constant(Cκ,Cλ)were obtained using pComb3XTT and pComb3X>as templates.The second round PCR amplification of heavy chain(Fd),light chains(κ and λ)was finished by splicing overlap extension(SOE-PCR).The complete Fab gene was obtained by SOE-PCR using the second round amplified products as the template.After inserting into pComb3XSS phage plasmid,a phage display Fab fragment of antibody library against SFTSV was constructed.Results The antibody Fab fragment sequences were amplified by three rounds of PCR,and a phage display antibody library with storage capacity of 1.875×10^(9)was successfully constructed.Conclusion Aphagedisplay antibody library was successfully constructed using blood specimens from SFTS convalescent patients with cross-neutralizing antibodies,which provided a reliable basis for further screening of specific neutralizing antibodies.