| 王巧,陈芝喜.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.[J].中南医学科学杂志.,2023,(6):829-833. |
| 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 |
| 投稿时间：2022-10-10 修订日期：2023-08-23 |
| DOI：10.15972/j.cnki.43-1509/r.2023.06.007 |
| 中文关键词: 冠状病毒 核衣壳蛋白 生物信息学 免疫学性质 [ |
| 英文关键词:coronavirus nucleocapsid protein bioinformatics immunological properties |
| 基金项目:湖南省自然科学基金(2021JJ70115)；衡阳市科技局项目(2020hcjz6717) |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| AimTo analyze the biological properties, purification and polyclonal antibody preparation of nucleocapsid (N) protein of novel coronavirus (SARS-CoV-2). MethodsThe 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. ResultsThe 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 of α-helix was 21.24%, β-sheet was 16.71 and β-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. ConclusionThe SARS-CoV-2 N protein contains a large number of intrinsic disorder domain and the high titer polyclonal antibody was obtained. |
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