新型冠状病毒S1蛋白的表达及活性鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2021-1076

摘要/Abstract

摘要：

旨在获得具有免疫活性的SARS-CoV-2 S1蛋白,为COVID-19的免疫预防及临床诊断提供较好的候选抗原。该研究利用PCR技术扩增出刺突蛋白（Spike,S）的S1亚基序列,通过同源重组法将目的片段克隆至冷休克表达载体pCold I,转化至大肠杆菌BL21感受态细胞内诱导表达S1蛋白,经Ni²⁺柱亲和层析纯化后,通过SDS-PAGE和Western blotting鉴定目的蛋白的表达情况。结果表明,已成功表达含His标签的S1蛋白,并且已获得浓度和纯度较高、能与S1多克隆抗体反应的目的蛋白。此外,蛋白刺激细胞的结果表明,原核表达的S1蛋白免疫原性良好,能诱导小鼠巨噬细胞上调表达多种细胞因子和趋化因子,促进RAW264.7细胞产生免疫反应。综上,该研究成功构建了pCold I-S1重组质粒,获得了SARS-CoV-2的良好候选抗原S1蛋白。

关键词: SARS-CoV-2, S1蛋白, 原核表达, 生物活性鉴定

Abstract:

This work aims to obtain SARS-CoV-2 S1 protein with high immunoactivity,and to provide a good candidate antigen for the immune prevention and clinical diagnosis of COVID-19. PCR was adapted to amplify the S1 subunit of S protein,and the homologous recombination to clone the target sequence into the cold shock expression vector pCold I,then the recombinant plasmid was transformed into competent cells Escherichia coli BL21 and induced to express S1 protein. After purification by Ni ²⁺column affinity chromatography,SDS-PAGE and Western blotting were used to identify the expressions of the target protein. The results showed that the S1 protein containing His tag was expressed successfully,also,S1 protein with high concentration and purity was obtained,which reacted with S1 polyclonal antibody. In addition,the results of cell stimulation by protein demonstrated that S1 protein by prokaryotic expression presented promising immunogenicity,and it significantly increased the expression of cytokines and chemokines in mice macrophages,thus promoting the immune response of RAW264.7 cells. In sum,this study successfully constructed the recombinant plasmid pCold I-S1,and obtained S1 protein that could be a good candidate antigen for SARS-COV-2.

Key words: SARS-CoV-2, S1 protein, prokaryotic expression, bioactivity identification

汪巧菊, 胡雨萌, 温亚亚, 宋丽, 孟闯, 潘志明, 焦新安. 新型冠状病毒S1蛋白的表达及活性鉴定[J]. 生物技术通报, 2022, 38(3): 157-163.

WANG Qiao-ju, HU Yu-meng, WEN Ya-ya, SONG Li, MENG Chuang, PAN Zhi-ming, JIAO Xin-an. Expression and Activity Identification of SARS-CoV-2 S1 Protein[J]. Biotechnology Bulletin, 2022, 38(3): 157-163.

图/表 7

表1 引物序列

Table 1 Primer sequence

基因 Gene	引物序列 Primer sequence（5'-3'）	产物大小 Product size/bp
IL-1β	F：GCCCATCCTCTGTGACTCAT	230
IL-1β	R：AGGCCACAGGTATTTTGTCG	230
TNF-α	F：AGCCCCCAGTCTGTATCCTT	212
TNF-α	R：CTCCCTTTGCAGAACTCAGG	212
MIP-2	F：AAGTTTGCCTTGACCCTGAA	180
MIP-2	R：AGGCACATCAGGTACGATCC	180
IP-10	F：GGATGGCTGTCCTAGCTCTG	211
IP-10	R：ATAACCCCTTGGGAAGATGG	211
β-actin	F：AGCCATGTACGTAGCCATCC	228
β-actin	R：CTCTCAGCTGTGGTGGTGAA	228

图1 S1基因PCR产物电泳分析 M：DL5000 DNA marker;1-2：S1基因PCR扩增产物

Fig. 1 Electrophoresis analysis of S1 gene PCR product M：DL5000 DNA marker. 1-2：PCR product of S1 gene

图2 重组质粒pCold I-S1的PCR鉴定 M：DL5000 DNA marker;1-2：pCold I-S1质粒的PCR扩增产物

Fig. 2 PCR identification of recombinant plasmid pCold I-S1 M：DL5000 DNA marker;1-2：PCR product of pCold I-S1

图3 S1蛋白的SDS-PAGE分析 M：Protein marker;1：BL21（pColdⅠ）诱导菌;2：BL21（pColdⅠ-S1）未诱导菌;3：BL21（pColdⅠ-S1）诱导上清;4：BL21（pColdⅠ-S1）诱导沉淀

Fig. 3 SDS-PAGE analysis of S1 protein M：Protein marker. 1：Induced BL21（pColdⅠ）. 2：Non-induced BL21（pColdⅠ-S1）. 3：Supernatant of induced BL21（pColdⅠ-S1）. 4：Precipi-tation of induced BL21（pColdⅠ-S1）

图4 纯化后S1蛋白的SDS-PAGE分析 M：Protein marker;1：BL21（pCold Ⅰ）诱导菌;2：BL21（pColdⅠ-S1）诱导上清;3：BL21（pColdⅠ-S1）诱导沉淀;4-5：纯化后S1蛋白

Fig. 4 SDS-PAGE analysis of purified S1 protein M：Protein marker. 1：Induced BL21（pColdⅠ）. 2：Supernatant of induced BL21 （pColdⅠ-S1）. 3：Precipitation of induced BL21（pColdⅠ-S1）. 4-5：The purified S1 protein

图5 纯化后S1蛋白的Western blotting鉴定 M：Protein marker;1：BL21（pColdⅠ）诱导菌;2：纯化后S1蛋白

Fig. 5 Western blotting identification of purified S1 protein M：Protein marker. 1：Induced BL21（pColdⅠ）. 2：The purified S1 protein

图6 S1蛋白刺激RAW264.7细胞后的细胞因子及趋化因子表达水平

Fig. 6 Expression levels of cytokines and chemokines in RAW264.7 cells stimulated by S1 protein

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