Heterologous expression of Cauliflower Mosaic Virus coat protein gene in E. coli and formation of virus-like particles
2023
Pouresmaeil, Mahin | Pazhouhandeh, Maghsoud | Shirzad, Akbar | Yari Khosroushahi, Ahmad
Cauliflower Mosaic Virus (CaMV) is the first identified plant virus with a DNA genome and causes great economic damage to Brassicaceae plants. The successful production of capsids of this virus is useful both in its serological diagnosis and in the formation of virus-like particles (VLPs). In this research, DNA from cauliflower samples with suspected virus symptoms was extracted and the coat protein (CP) gene of the virus was amplified using specific primers in PCR. Then it was inserted into the expression vector pGEX2TK downstream of GST by digestion endonuclease enzymes BamHI and SmaI and after confirmation of the recombinant vector, its sequencing was carried out. Sequencing was performed and the sequence obtained in the NCBI database was compared with the sequences in the gene bank with the BLASTn program. It was found that this sequence is related to the CaMV virus with accession number KF357590.1 in the NCBI database. The expression of the CP was induced and optimized in E. coli (Rosetta strain). A very high expression of this protein was obtained at 37°C and 6 hours after induction using IPTG with a final concentration of 1 mM. Then, this protein was purified with sepharose nanoparticles which have an affinity to GST, and observed in SDS-PAGE. The related electron microscope images confirm the production of VLPs of this virus with correct assembly without creating inclusion bodies that can be tested in drug delivery in medicine.
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