SARS-CoV-2 N蛋白不同片段制备及其在荧光层析中的应用

Preparation of different fragments of SARS-CoV-2 N protein and its application in fluorescence chromatography

摘要:

表达纯化新型冠状病毒核衣壳 (N) 蛋白不同片段，建立新冠病毒总抗体荧光免疫层析方法并评价不同蛋白片段对该方法的影响。利用生物信息学技术对N蛋白序列进行分析、合成及原核表达、纯化，制备不同N蛋白片段；采用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺 (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride，EDC) 法荧光微球偶联抗原建立夹心荧光层析抗体检测方法，并分别进行性能评价。在制备的4个N蛋白片段中优选出全长N蛋白 (N419) 包被，N412加入0.5 mol/L NaCl进行标记作为最优组合；删掉N抗原N端第91–120位氨基酸 (N412) 可减少87.5%的非特异性干扰；线性范围为0.312–80 U/L，最低检出限为0.165 U/L，准确度在95%以上。优选配对N蛋白片段建立的新冠病毒总抗体荧光免疫层析检测方法，与广州万孚试纸条对比总符合率为98%，有望用于新型冠状病毒的辅助检测，同时为新冠抗体检测试剂性能的提升提供实验依据和借鉴。

Abstract:

Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91–120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312–80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.