Preparation of nanobody against N protein of SARS-CoV-2 and assembly of colloidal gold immunochromatography card
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摘要:
目的 通过制备新冠病毒N蛋白纳米抗体,将其应用于新冠病毒N蛋白抗原检测。 方法 将新冠病毒N蛋白免疫羊驼,构建N蛋白纳米抗体基因文库,通过噬菌体展示技术淘筛对新冠病毒N蛋白具有亲和力的纳米抗体,将纳米抗体基因克隆到酵母载体,挑取阳性克隆菌株诱导表达,亲和层析纯化分泌蛋白,透析脱盐和浓缩后,采用ELISA法筛选对新冠病毒N蛋白高亲和性纳米抗体,交叉配对筛选出1对纳米抗体制备成胶体金免疫层析检测卡,滴加N蛋白溶液到样品孔观察检测线和质控线。 结果 共筛选出46株不同序列的纳米抗体,优选其中6株表达、纯化和浓缩得到纳米抗体,抗体浓度 > 1.0 mg/mL。ELISA筛选结果显示,6株纳米抗体均对N蛋白有高亲和力,抗体配对实验筛选到1对抗体可以夹心法检测新冠病毒N蛋白,组装成胶体金免疫层析检测卡,对新冠病毒N蛋白抗原检测可见明显检测线和质控线条带,检测限 ≥ 10 μg/mL。 结论 制备的纳米抗体可以用于胶体金纳米抗体免疫层析检测卡,实现对新冠病毒N蛋白的检测。 Abstract:Objective To prepare nanobodies against N protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and apply them to the detection of N protein antigen of SARS-COV-2. Methods The alpaca was immunized with N protein to construct a gene library of nanobody to N protein. The nanobody with affinity to N protein was screened with phage display technology; nanobody genes were cloned to yeast plasmids; positive clone strains were picked to induce expression; the secreted proteins were purified with affinity chromatography, desalted with dialysis and concentrated, then screened with enzyme-linked immunosorbent assay (ELISA) for the collection of high affinity nanobodies to N protein. A pair of nanobodies was screened with cross pairing experiment for the preparation of colloidal gold immunochromatography detection cards, and N-protein solution was dropwise added to the sample wells to observe the detection line and quality control line. Results Totally 46 nanobodies with different sequences were screened out and 6 of them were selected to express nanobodies with the concentration > 1.0 mg/ml and high affinity to N protein after purification, desalting and enrichment. Finally, the selected pair of antibodies was assembled into colloidal gold N protein immunodetection card to detect N protein with sandwich method. Obvious detection line and quality control line were observed in the test of SARS-CoV-2 N protein antigen using the detection card and the detection limit was ≥ 10 μg/mL for the test. Conclusion The prepared nanobody can be used in the colloidal gold nanobody immunochromatography detection card to detect SARS-CoV-2 N protein. -
表 1 纳米抗体噬菌体展示库筛选结果
筛选轮数 输入效价
(pfu)筛选效价
(pfu)对照效价
(pfu)富集程度 相差倍数 第1轮 1 × 1012 4.4 × 108 2.4 × 106 4.4 × 10–4 183 第2轮 5.2 × 1011 2.4 × 108 1.6 × 106 4.1 × 10–4 150 第3轮 8.4 × 1011 1.6 × 109 2.4 × 106 1.9 × 10–3 667 表 2 ELISA检测纳米抗体对抗原N蛋白和阴性对照BSA的吸光值
编号 1 2 3 4 5 6 N蛋白 1.227 1.436 1.298 0.601 0.985 0.455 BSA 0.123 0.145 0.073 0.081 0.084 0.076 -
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