猪圆环病毒2型特异性纳米抗体文库的构建及鉴定

Construction and Characterization of Specific Nanobody Library against PCV2

关键词:  猪圆环病毒2型  羊驼  纳米抗体文库  抗体库容量  多样性

Key words:  PCV2  Alpaca  Nanobody library  capacity of antibody library  diversity

摘要:

为构建猪圆环病毒2型(PCV2)特异性纳米抗体文库，并对其库容和多样性进行鉴定分析，使用PCV2全病毒灭活疫苗免疫羊驼，经四次免疫后采血分离外周血淋巴细胞，提取总RNA，通过RT-PCR反转录合成cDNA；根据羊驼重链抗体基因序列设计特异性引物，通过巢式PCR扩增羊驼单域重链抗体(variable domain of heavy chain of heavy-chain antibody，VHH)基因片段vhh，并将其克隆到噬菌粒载体pCANTAB 5E中，构建pCANTAB- vhh重组质粒；通过电穿孔法将重组质粒导入E. coli TG1获得抗PCV2特异性纳米抗体基因文库。文库菌梯度稀释后计数单克隆菌落数，并通过菌液PCR鉴定重组菌阳性率，计算抗体文库库容量；对PCR鉴定为阳性的重组质粒进行测序，分析抗体文库多样性。结果显示，构建的PCV2特异性纳米抗体文库重组菌阳性率为92.0%，库容量为2.75×10¹⁰ CFU/mL；测序后序列比对分析结果表明，纳米抗体文库包含的基因序列具有丰富的多样性。本研究成功构建了PCV2特异性纳米抗体文库，且文库质量满足后续试验要求，为进一步获得针对PCV2抗原的纳米抗体奠定了基础。

Abstract:

To construct specific nanobody library against PCV2 and to characterize its capacity and diversity, a female alpaca was immunized with inactivated PCV2. Anticoagulated blood was collected after the fourth immunization, and peripheral blood lymphocyte was separated, followed by extraction of total RNA, and then cDNA was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) method. Specific primers were designed according to literature data and heavy chain antibody gene sequences of alpaca published in GenBank and were used to amplify variable domain of heavy chain of heavy-chain antibody gene segment (vhh) by nested PCR. And then the vhh segment was cloned into phagemid vector pCANTAB 5E to construct pCANTAB-vhh recombinant plasmid, which was then transformed into E. coli TG1 by electroporation. For calculating the capacity of the nanobody library against PCV2, library bacteria monoclonal colony number was counted after gradient dilution and positive rate of library bacteria was identified by PCR method. Positive recombinant plasmid was extracted and was sequenced. The results showed that the positive rate of antibody library recombinant bacteria was 92.0% and the nanobody library capacity was 2.75×10¹⁰ CFU/mL. Sequences alignment analysis results indicated that the nanobody library contained abundant diversity of gene sequences. This study successfully constructed a specific nanobody library against PCV2 and the quality of the library met the requirement of subsequent research, which made it possible for further obtaining high affinity nanobody against PCV2.