- 中文核心期刊
- CSCD来源期刊
- 中国科技核心期刊
- CA、CABI、ZR收录期刊
| Citation: |
LIN E S. Expression and Characterization of Capsid Protein of Eel Circovirus [J]. Fujian Journal of Agricultural Sciences，2024，39（11）：1236−1241. DOI: 10.19303/j.issn.1008-0384.2024.11.004
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The capsid protein (Cap) of Eel circovirus (EeCV) was prepared using a prokaryotic expression system for the development of an EeCV subunit vaccine and related biological products.
The genome sequence of EeCV-Cap (GenBank accession number: NC_023421.1) was used as the reference for codon optimization and plasmid synthesis. The Cap gene was amplified with designed specific primers and cloned into pET-32a vector to construct the pET32a-EeCV-Cap recombinant expression plasmid. EeCV-Cap recombinant protein was obtained by transforming the recombinant plasmid into the host bacterium BL21 (DE3) followed by an IPTG induction. Western blot was performed to confirm expression of the recombinant Cap.
The recombinant pET32a-EeCV-Cap was mainly expressed as a soluble protein in E. coli. A purified target protein was obtained using nickel affinity chromatography with the highest expression achieved with the final IPTG concentration of 0.5 mmol·L−1 for the induction at 20 ℃ in 16 h. A single band with the calculated molecular weight of 31 kDa was detected by Western blot. Under a transmission electron microscope, the negative phosphotungstic acid-stained protein suspension showed numerous regular virus-like particles (VLPs) that were 20 nm in diameter.
The solubility expression of EeCV-Cap in E. coli was realized in this study to visualize the VLPs. Using the modified nickel affinity chromatography, a single target protein was obtained that could spontaneously assemble into VLPs in vitro for the development of an EeCV subunit vaccine and related products.
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