Abstract
Here we described the cloning, bioinformatic characterization and expression in Escherichia coli of the major capsid protein (MCP) from marine unicellular algae Emiliania huxleyi virus EhV-99B1 isolate. The purified recombinant MCP was used to develop a polyclonal antibody for testing viral infection. The full length open-reading frame (ORF) of MCP encodes a protein of 496 amino acids with a calculated molecular mass of 55 kDa and Ip 6.34. Hydropathy analysis of MCP showed that there were 6 largely hydrophobic domains, which may be important for the interaction with the envelope protein. The conserved region of EhV strains MCP had high similarity in amino acid sequence and secondary structure which allow us to develop a specific biomarker for EhVs infection detection. The full length ORF was subcloned into expression vector pGEX-4T-3 for overexpression in E. coli as glutathione-Stransferase-L1 (GST-L1) fusion protein and the soluble recombinant protein was used to generate polyclonal antibodies in mice. The obtained antisera reacted in Western immunoblots with the same protein both in purified EhV-99B1 virions and infected host cells sample. These shows that the antiserum against recombinant EhV-MCP offers the potential to develop immunofluorescence techniques for the detection of EhVs infected cells.
Highlights
Characterization and expression in Escherichia which burst size must be known.5 An existing Conflict of interests: the authors declare no coli of the major capsid protein (MCP) from marine unicellular algae Emiliania huxleyi virus ly E. huxleyi-specific viruses (EhV)-99B1 isolate
The full length openo reading frame (ORF) of MCP encodes a protein of 496 amino acids with a calculated molecular e mass of 55 kDa and Ip 6.34
The full length ORF was subcloned into expression e vector pGEX-4T-3 for overexpression in E. coli as m glutathione-Stransferase-L1 (GST-L1) fusion protein and the soluble recombinant protein was m used to generate polyclonal antibodies in mice
Summary
The soluble GST-MCP fusion protein was purified by PCR reaction mixture which contained KOD hydrophobic interaction chromatography. L Viral purification and DNA ia isolation c To produce purified virus, the virus was r amplified in 1.5L culture grown in f/2 medium e (Guillard, 1975). The PCR product was purified with agarose gel DNA purification kit Ver.2.0 (TaKaRa) and was cloned into the pUCM-T Vector System (Invitrogen) for sequence. To obtain a sufficient amount of soluble recombinant protein, MCP was overproduced in E. coli BL21 using the pGEX-4T-3 vector. In order to obtain more soluble recombinant protein, the recombinant strain, harboring the plasmid pGEX-MCP, was induced with different concentrations of IPTG (0.1 mM, 0.3 mM and 0.6 mM) at different temperatures (20, 25 and 30°C) for different induction durations (5 h, 8 h and 10 h) respectively. The purified fusion protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
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