| The human cytomegalo virus (HCMV) contains about 20~25 proteins. Among these, there are three proteins: phosphoprotein pp71(encoded by ORF UL82), pp65(encoded by ORF UL83)and pUL69(encoded by ORF UL69)play an important role in the process of CMV-mediated infection and viral replication. One common characteristic of the three tegument proteins is that they can activate host cell infection and viral replication, meanwhile; they also play an important part in sneaking through by T lymphocyte-mediated cytotoxicity. Recent research has indicated that pp71 encoded by ORF UL82 is of the most significance among the three tegument proteins. Presenting alone, pp71 protein is a stronger protein antigen and it may stimulate organism to generate antibodies, which will be helpful to clean HCMV. To 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 the degradation of oncoproteins p105, p107, p130 etc. The pp71 protein can induce resting cells to enter into cell cycle and then keep them resting in later G1 phase, which is the advantageous phase for viral replication. So it can 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 increase 80 times. Studies have 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. Thus, Baldick pointed out that ORF UL82(pp71 protein)might take for effective target dot used in the design of anti-CMV drug. In order to further investigate the expression of pp71 protein gene in eukaryotic cell—human foreskin fibroblast, and establish scientific basis for setting up and screening on humanized gene engineered antibody bank of HCMV pp71, following jobs have been done in this study: 1. Cultivation of human foreskin fibroblast. Inoculating the third generation frozen HFF into DMEM complete medium with 10% fetal bovine serum, the next generation cells, which have been divided into 1:3 will be cultivated in 37℃,5%CO2 incubation cabinet when they grow enough to get close to culture flask wall, will be marked with the generation time,name and so on. After propagating the fourteenth generation, these logarithmic phase cells are frozen to use in the following experiments. 2. Assessment of genetic recombinant plasmid pSG5-pp71. (1) Transfer recombinant plasmid pSG5-pp71 into DH5αE.coli by using the method of Calcium Chloride. (2) Extract recombinant plasmid pSG5-pp71and then detect elementarily the positive cloning gene via single enzyme and double enzyme digestion. Design primers: by using the method of PCR to expand pp71 target gene fragments for further detection, and then to sequence. Comparing the sequencing results of DNA fragment of pp71 gene with standard sequence in gene bank, homology is 99.82% and therefore it means the construction of recombinant plasmid get success. 3.Expression ,purification and identification of recombinant plasmid in HFF. (1)Stable transfection of recombinant plasmid pSG5-pp71 in human foreskin fibroblast. (2)To detect the recombinant plasmid pSG5-pp71 by RT-PCR by using the total RNA extracted from HFF as the template and taking two couples of synthetic primers as the amplification object. (3)Identification of gene expression product: the lysate of human foreskin fibroblast expressed gene recombinant plasmid pSG5-pp71 is firstly purified by Ni-NTA affinity chromatograph and then separate from SDS-PAGE protein electrophoresis and Western blot finds that a specific strap emerges in molecular weight about 71 000, which aims directly at pp71 antibody. This is identical with molecular weight in theory.
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