Cloning And Expression Of Human Cytomegalorvirus (HCMV) PP65 In E.coli And Studies On The Immunigenicity Of The Recombinant

The human cytomegalovirus (HCMV) contains about 20~25 proteins, in respect of HCMV-mediated infection and viral replication, there are 3 most important proteins, pp65 (encoded by ORF UL83), phosphoprotein pp71 (encoded by ORF UL82), and pUL69 (encoded by ORF UL69). One common characteristic of the three all tegument proteins is to active host cell infection and viral replication, while they play an important role to immunize from T lymphocyte-mediated cytotoxicity. Recent studies show that pp65 encoded by ORF UL83 has the most significant characteristic among the three tegument proteins.Presenting alone, pp65 protein is a stronger protein antigen, it may stimulate organism to generate antibodies, thus will be helpful to clean HCMV; For HCMV infection of transplant recipients, the generation of pp65 protein may significantly enhance expression of intercellular adhesion molecule-1 (ICAM-1), therefore it will cause allograft vasculopathy and rejection; For retinoblastoma, the expression of pp65 protein may cause degradation of oncoproteins p105, p107, pl30 etc. The pp65 protein may induce resting cells enter into cell cycle and rest in later G1 phase, which is the advantageous phase for viral replication, so it could increase viral reproduction, speed viral transfer between cells. After human fibroblast was transfected by CMV in vitro, with cotransfection of ORF UL83(pp65 protein), viral infection and DNA replication efficiency increases 80 times. Studies proved that pp65 has promoting influence not only on IE1 and IE2 gene expression of HCMV, but also on late gene expression, as well as promoting and enhancing influence on viral transfer to normal host cell. So Baldick pointed out that ORF UL83 (pp65 protein) may take for effective target dot used in the design of anti-CMV drug, following jobs have been done in this study:1. Cloning pp65 gene of HCMV and constructing pET32-pp65 expression plasmid:(1)Extract HCMV DNA using proteinase K.(2)Design primers for PCR to amplify the pp65 target gene fragment.