Molecular Cloning, Expression And Application Of The Two Functional Genes From Marine Emiliania Huxleyi Virus

Viruses are the most abundant and genetically diverse ‘life forms’ in the ocean, typicallythere are106～109VLPs/ml. Not only are viruses abundant in oceans but, as is becoming clear,they also harbor enormous genetic and biological diversity. Of the large number, more than50%conding sequences （CDSs） haven’t been annotated. Genomic sequence analysis of Emilianiahuxleyi virus （Coccolithovirus, EhV） makes it the largest Phycodnaviridae genome sequenced todate. It encoded a large proportion of CDSs and showed no significant similarity to any otherDNA or protein sequences in existing databases. The absence of these sequences from publicdatabases may indicate a specific role for these proteins in EhV or the interaction between EhVand its host. In this study we described the cloning, bioinformatic characterization andexpression in Escherichia coli of the major capsid protein （MCP） and thioredoxin （Trx） fromEhV-99B1isolate. The recombinant Trx was used to minimize the allergen of food, while therecombinant MCP was used to generate specific antibody and value a potential application ofMCP as a diagnostic marker for E. huxleyi-specific viral infection.The Trx gene of EhV-99B1was amplified by PCR with specific primers and subcloned intothe prokaryotic expression vector pGEX-4T-3to get the recombinant plasmidpGEX-4T-3/EhV-99B1-Trx. A positive plasmid was transformed into the host cell Origami andthe target gene was successfully expressed. The fusion protein was purified by GST-Sepharose4B chromatography and the activity of disulfide reductase was detected. Second andthree-dimensional structures of EhV-99B1Trx are predicted by the tools in bioinformatics. Theresults showed that:（1） The Trx gene had a open reading frame of591bp, which contained adeduce amino acid sequence of196residues with a calculated molecular mass of22.1kDa andits theoretical pI was5.27; subcellular localization suggested that Trx may be a endoplasmicreticulum product protein;（2） A conserved thioredoxin-like domain was detected in theN-terminal region of EhV-99B1Trx, with a typical conserved Cys-Gly-Pro-Cys （CGPC） residuemotif active site;（3） Phylogenetic relationship between EhV99B1-Trx and homologues fromother organisms are evaluated, indicating a high identification with known CoccolithovirusEhV-86Trx gene （NC₀07346） in98%and100%of nucleotide and deduced amino acidsequences, respectively. However, EhV-99B1Trx has only about9.8%～18.8%identity inamino acid sequence with other organisms;（4） An analysis of the predicted second and3-dimensional structural modeling and folding pattern further suggested that EhV-99B1Trx similar to Trx2, about24.76%identity in amino acid sequence with Homo sapiens Trx2andEhV-99B1-Trx-like is identifiable as a new member of thioredoxin super family;（5） Thepredicted functions of EhV-99B1Trx are related to cell envelope, stress response and immuneresponse;（6） As found for other proteins with intramolecular disulfide bonds, β-lactoglobulinand insulin were reduced specifically by the recombinant EhV-99B1Trx and the treated productwas relatively stable, which indicated that the EhV-99B1Trx, as a new kind of thioredoxin, dohave the potential in food safety areas. It also will lay a foundation for the futher study in thestructure and function of EhV-99B1Trx, and the interactional mechanisms between EhV and itshost.Here we describe the cloning, nucleotide sequencing and expression in E. coli of the MCPfrom EhV-99B1isolate. The MCP gene was subcloned into the prokaryotic expression vectorpGEX-4T-3to get the recombinant plasmid pGEX-4T-3/EhV-99B1MCP. A positive plasmidwas transformed into the host cell BL21and the target gene was successfully expressed. Thefusion protein was purified by electroelution. In addition, bioinformatics approach was utilizedfor analyzing EhV-99B1MCP major epitopes. Using overlap extension PCR technique forepitope gene splicing and subcloned into the prokaryotic expression vector pET28α+foroverexpression in E. coli. The fusion protein was purified by His-tag chromatography. Thepolyclonal antibody and monoclonal antibody against MCP were performed by immunizing theBABL/c mice. The results showed that:（1） The full open-reading frame （ORF） of MCPencoded a protein of497amino acids with high hydrophobicity, calculated molecular mass of55kDa and theoretical pI of6.34; subcellular localization indicated that MCP may be a cytosolicprotein;（2） A BLAST P search indicated that there is about93%～100%identity in theconserved domain of nucleotide and100%deduced amino acid sequence with other EhV isolates,which suggests that EhV MCP might have been conserved throughout this family. Based uponthe sequence homology and the high conserved secondary structural domain among EhV isolates,it could be expected that the antibody generate from MCP could serve as a specific marker forthe detection of EhVs infected cells;（3） Hydropathy analysis of EhV-99B1MCP indicated thatthe regions from N-to C-terminal residues43H-60Q;74D-94A;232L-288V;344N-366K;402P-431S;470L-493N were largely hydrophobic, which may be important for the interactionwith the envelope protein;（4） The MCP amino sequence has two O-glycosylation sites, severalphosphorylation sites and two caspases recognizes highly specific tetrapeptide motifs（PDFD-172to176&SRLD-451to454） of N-terminal;（5） Antisera raised against purifiedEhV-99B1MCP major epitopes fusion protein reacted specifically with purified EhV-99B1inWestern immunoblots. These shows that the purified recombinant EhV MCP major epitopescould be useful for further research in its characteristics, functions, viral susceptibility and viral assembly in vitro, and the antiserum against recombinant EhV-MCP offers the potential todevelop immunofluorescence techniques for the detection of EhVs infected cells. It is helpful forvaluing EhV mediated death in marine environment.