Cloning And Expression Of Human Cytomegavirus UL82 Gene In E.coli

The human cytomegalovirus (HCMV) contains about 20～25 proteins, in respect of CMV-mediated infection and viral replication, there are 3 most important proteins, phosphoprotein pp71 (encoded by ORF UL82), pp65 (encoded by ORF UL83) and pUL69 (encoded by ORF UL69). One common characteristic of the three all tegument proteins is activate host cell infection and viral replication, while they play an important role to immunize from T lymphocyte-mediated cytotoxicity. Recent studies show that pp71 encoded by ORF UL82 has the most significant characteristic among the three tegument proteins. Presenting alone, pp71 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 pp71 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 pp71 protein may cause degradation of oncoproteins p105, p107, p130 etc. pp71 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 UL82 (pp71 protein), viral infection and DNA replication efficiency increases 80 times. Studies proved that pp71 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 UL82 (pp71 protein) may take for effective target dot used in the design of anti-CMV drug.In order to further research the expression of pp71 gene in E.coli and effect on purified protein in viral reproduction, and provide scientific basis for research on CMV's pathogenicity, diagnoses and therapy, following jobs have been done in this study:Constructing HCMV infection platform Inoculating HCMV into HFF in DMEM culture with 2% calf serum, harvesting cells until most cells obviously appear CPE and storage under –70℃. The detection of pp65 antigen in cultured cells using immunohistochemistry shows that most of cells were infected by HCMV successfully.Cloning pp71 gene of HCMV and constructing pGEX-4T-1 expression plasmidExtract HCMV DNA using proteinase K.Design primers for PCR to amplify the pp71 target gene fragment.Using BamHI和EcoRI double digest target fragment and pGEX-4T-1. Connecting pp71 target gene fragment with pGEX-4T-1 and transfer recombinant plasmid pGEX/pp71 into DH5a using the method of Calcium chloride.Extract recombinant plasmid pGEX/pp71, detect the recombinant plasmid via double digestion and PCR, then send it to company to sequence it. Comparing the sequencing results of DNA fragment of pp71 gene with standard sequence in gene bank, homology is 99.6%, it means the construction of recombinant plasmid get success. Expression of pGEX/pp71 recombinant plasmid in E.coli. Using inducement of IPTG to express target protein effectively, the expression account for about 30% of total protein. Its molecular weight is the same as theoretical molecular weight of 71KD.