Construction Of Human Cytomegalovirus Genomic Expression Library To Study Interactions Between HCMV Encoding Proteins

Objects:In order to provide an effective tool for further studying of the function and regulatory mechanism of HCMV encoding proteins, a random genomic library of HCMV strains Towne fused to the GAL4 activation domain, was constructed. Using this library, we performed yeast two-hybrid system to select HCMV encoding proteins interacting with HCMV tegument protein pUL23 which encoded by UL23 gene. These research will be laid a foundation to elucidate the functions of pUL23 during HCMV infection.Methods:1) Construct the random genomic library of HCMV:The HCMV Towne-BAC DNA was extracted and cleavaged by DNasel to get 0.5-2.5kb random DNA fragments. The DNA fragments were blunt ended by T4 DNA polymerase and then added A-Tail. A-Tailed DNA fragments connected to the vector of pMD18T plasmid by T-A cloning and then transformate E. coli DH5 a. The random genomic library of HCMV—pGADT7-HCMV was constructed by double enzyme cutting technique and the E. coli DH5αwas transformated.2) Yeast two-hybrid assay: Yeast AH109 was cotransformated with the genomic library pGADT7-HCMV and the bait plasmid pGBKT7-UL23. Clones were picked out forβ-galactosidase assay. Then the plasmid of positive clones were extracted from yeast and rescued via electriotransformation of E. coli DH5α. The unique clones were delivered to sequenceing, and BLAST in GenBank.3) GST-pull down assay:Recombinant procaryotic expression plasmid pGEX4T-1-UL24 was constructed and transformated to the Tuner(DE3) to produce the GST-UL24 fusion protein by IPTG induction. The plasmid pcDAN3.1(+)-Flag-UL23 was constructed and expressed in cos-7 cells.Western Blotting with Anti-Flag antibody was excuted after the coincubation of GST-UL24 and Flag-UL23 proteins.4) Co-immunoprecipitation assay:The plasmid pcDAN3.1(+)-HA-UL24 was constructed and cotransfected into cos-7 cells with pcDNA3.1(+)-Flag-UL23. After collecting the supernatant of cell lysate and being immunoprecipitated by Anti-HA antibody, fusion proteins of HA-UL24 and Flag-UL23 were detected by Western blotting with Anti-HA and Anti-Flag antibodies, respectively.Results:Half of 6 positive clones which were gained in the yeast two-hybrid experiment belong to HCMV. One of the interacting clones corresponds to HCMV protein pUL24. GST-pull down and Co-immunoprecipitation assays, as well yeast two-hybrid test, confirmed the interaction between these two viral proteins.Conclusions:These results demonstrated that the viral genomic library could be applied to GAL4 yeast two-hybrid assay for screening HCMV encoding proteins which interact with HCMV pUL23. And the interaction between HCMV viral proteins will be further characterized to elucidate the functions of pUL23 during HCMV infection. It was laid a foundation for further studying of the infection mechanism of HCMV.