牛冠状病毒N蛋白抗体库构建与单链抗体筛选

Construction of Phage Display Antibody Library and Screening of Single Chain Antibody against Bovine Coronavirus N Protein

为快速筛选获得牛冠状病毒(BCoV)特异性抗体,本试验用重组的牛冠状病毒N蛋白免疫BALB/c小鼠,从免疫小鼠脾细胞中提取总RNA,反转录成cDNA后,分别扩增抗体重链可变区(VH)和轻链可变区(VL)基因,采用重叠延伸PCR法将VH、VL拼接为完整的单链抗体(scFv)基因,将scFv基因与酶切的噬菌粒连接,电转入感受态细胞中,过夜培养获得初级噬菌体抗体库,测定库容为8×10^(8)PFU。自库中随机挑取15个单克隆的噬菌体通过PCR鉴定,结果显示其中13个克隆扩增出约800 bp大小的目的片段,阳性率为86.7%,说明噬菌体抗体库构建成功。该抗体库经4轮富集淘选和酶联免疫吸附试验检测,最终获得32株与N蛋白特异性反应的噬菌体克隆。将32株阳性克隆进行PCR鉴定并测序,分析序列后发现有19株阳性单链抗体基因正确,选取阳性值较高的5株阳性单链抗体检测其噬菌体抗体结合活性,其中阳性单链抗体scFv-154与N蛋白的结合活性较高。本试验为进一步探讨单链抗体在牛冠状病毒病诊断和防控技术领域的应用提供了依据。

To quickly screen for bovine coronavirus(BCoV)-specific antibodies,this study utilized recombinant bovine coronavirus N protein to immune BALB/c mice.After reverse transcription of total RNA from splenocytes of immunized mouse into cDNA,the antibody heavy chain variable region(VH)and light chain variable region(VL)genes were amplified,respectively,and ligated to a complete single chain antibody(scFv)gene by overlapping extension PCR.scFv gene was ligated to the digested phagemid and electro-transformed into competent cells.Primary phage antibody library was obtained by overnight culture and the capacity was 8×108 PFU,and 15 monoclonal phages were randomly selected from the library for PCR identification.The results showed that 13(86.7%)of the 15 monoclonal phages contained a target fragment of about 800 bp,indicating the successful construction of the phage antibody library.Following 4 rounds of enrichment and enzyme-linked immunosorbent assay(ELISA),32 higher positive scFv were obtained,of which 19 positive scFv genes had correct sequences.Five positive scFv with higher positive values were assessed for phage antibody binding activity,with scFv-154 showing high reactivity to N protein.The results from this study have provided a basis for further exploration of scFv in the diagnosis and prevention of bovine coronavirus disease.