Yee‑Huan Toha, Yu‑Weng Huangb, Yo‑Chen Changc, Yi‑Ting Chenb, Ya‑Ting Hsud, Guang‑Huey Lind,e*
aDepartment of Life Sciences, School of Medicine, Tzu Chi University, Hualien, Taiwan, bDepartment of Molecular Biology and Human Genetics, School of Medicine, Tzu Chi University, Hualien, Taiwan, cDepartment of Laboratory Medicine and Biotechnology, School of Medicine, Tzu Chi University, Hualien, Taiwan, dMaster Program in Microbiology and Immunology, School of Medicine, Tzu Chi University, Hualien, Taiwan, eInternational College, Tzu Chi University, Hualien, Taiwan
Open Access funded by Buddhist Compassion Relief Tzu Chi Foundation
Abstract
Objective: The coronavirus disease 2019 (COVID‑19) pandemic caused by the SARS‑CoV2 virus continues to pose a serious threat to public health worldwide. The development of rapid diagnostic kits can assist the Tzu Chi Foundation in supporting global volunteers working to provide relief during the current pandemic. Materials and Methods: In this study, nucleotide sequences derived from publicly available viral genome data for several domains of the SARS‑CoV2 spike and nucleocapsid (N) proteins were chemically synthesized, with codon optimization for Escherichia coli protein expression. No actual viral particles were involved in these experiments. The synthesized sequences were cloned into an E. coli expression system based on pQE80L, and expressed viral proteins were subsequently purified using Ni‑affinity chromatography. Western blotting was conducted using human antiviral sera to assess the response of codon‑modified viral proteins to COVID‑19 patient sera. Results: N protein was expressed in amounts large enough to support large‑scale production. The N‑terminal domain, receptor‑binding domain (RBD), Region 3, and the S2 domain were expressed in small but sufficient amounts for experiments. Immunoblotting results showed that anti‑N IgG and anti‑N IgM antibodies were detected in most patient sera, but only 60% of samples reacted with the recombinant RBD and S2 domain expressed by E. coli. Conclusion: The results indicated that codon‑optimized SARS‑CoV2 viral proteins can be expressed in E. coli and purified for rapid antibody detection kit preparation, with the codon‑optimized N protein, RBD, and S2 protein demonstrating the most potential.
Keywords: Codon optimization, COVID‑19, Escherichia coli expression system, N protein, SARS‑CoV2 antisera
