纳米抗体的稳定性及其结构基础研究进展

  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。

Virology,epidemiology, patho-genesis,and control of COVID-19

  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.