Expression and Activity Identification of SARS-CoV-2 S1 Protein

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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）

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

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

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

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