SARS-CoV-2核衣壳蛋白的生物信息学分析、纯化及多克隆抗体制备

Bioinformatics analysis,purification and immunological characterization of nucleocapsid protein of SARS-CoV-2

目的分析新型冠状病毒(SARS-CoV-2)核衣壳(N)蛋白的生物学性质、纯化及多克隆抗体制备。方法NCBI检索感染人的冠状病毒N蛋白氨基酸序列,采用Clustal Omega软件对其进行序列比对。利用SOPMA软件预测SARS-CoV-2 N蛋白的二级结构;应用Pondr和IUPred软件预测SARS-CoV-2 N蛋白的固有无序结构域;运用MoRFchib软件预测SARS-CoV-2 N蛋白的分子识别特征序列;利用IEDB网络分析其B细胞表位。纯化的N蛋白免疫BALB/c小鼠,分离小鼠血清,制备多克隆抗体;ELISA法测定血清中多克隆抗体滴度;MTT法检测N蛋白刺激小鼠脾细胞增殖情况。结果SARS-CoV-2 N蛋白与SARS-CoV N蛋白的同源性高于97%。N蛋白二级结构的无规卷曲占55.13%,α螺旋占21.24%,β折叠占16.71%,β转角占6.92%。N蛋白含有大量固有无序样结构,具有4个分子识别特征序列和11个线性B细胞表位。全长为51.5 kDa N蛋白可诱导BALB/c小鼠分泌高滴度抗体,并能导致淋巴细胞增殖。结论SARS-CoV-2 N蛋白含有大量固有无序结构域并获得了高滴度多克隆抗体。

Aim To analyze the biological properties,purification and polyclonal antibody preparation of nucleocapsid(N)protein of novel coronavirus(SARS-CoV-2).Methods The amino acid sequences of the N protein coronaviruses that can infect human beings were download from NCBI,and the sequences were aligned with Clustal Omega software.The secondary structure of SARS-CoV-2 N protein was predicted by SOPMA software.The intrinsic disorder domain of SARS-CoV-2 N protein was predicted by Pondr and IUPred,and the molecular recognition characteristic sequence of SARS-CoV-2 N protein was predicted by MoRFchib software;IEDB website was used to predict its B cell epitope.Purified N protein was used to immunize BALB/c mice,isolate mouse serum,and prepare polyclonal antibodies.Polyclonal antibody titer in serum was determined by ELISA.The proliferation of splenocytes stimulated by N protein was detected by MTT assay.Results The homology between SARS-CoV-2 N protein and SARS-CoV N protein was higher than 97%.The content of random coil is as high as 55.13%.The content ofα-helix was 21.24%,β-sheet was 16.71 andβ-turn was 6.92%.The SARS-CoV-2 N protein contains a large number of intrinsic disorder structures,with 4 molecular recognition characteristic sequences and 11 linear B cell epitopes.The purified N protein has a molecular weight of 51.5 kDa and high purity,which can stimulate mice to secrete high titer antibody and cause lymphocyte proliferation.Conclusion The SARS-CoV-2 N protein contains a large number of intrinsic disorder domain and the high titer polyclonal antibody was obtained.