| Human papillomaviruses (HPVs) are double-stranded covalentlyclosed circular DNA (cccDNA) viruses, and induce hyperproliferativelesions in epithelial tissues. To date, more than 100 genotypes of HPV havebeen identified and sequenced. HPVs can be divided into two groups,high-risk HPV types and low-risk HPV types, according to the benign ormalignant tumors they caused. High-risk HPVs, such as HPV16, 18, 31,induce lesions in the genital tract that can progress to malignancy. Theirgenomes will integrate into the chomosome of the host cell, and induceimmortalization and transformation. In contrast, low-risk HPV types, whichalso infect genital epithelia, primarily induce benign lesions, e.g.condyloma acuminatum. Considering the pathogenicity, a vast number of studies have beendone on high-risk HPVs, and the mechanism of high-risk HPVs inmalignancy has demonstrated. However, the roles of low-risk HPVs inbenign lesions are still unknown. Low-risk does not mean unimprotant. Inrecent years, with the rising incidence of diseases caused by low-risk HPVs,especially the most common type HPV11, study on them seems criticallyimportant and urgent. It has become increasingly clear that E6 proteins encoded by thehigh-risk HPV form a complex with p53 that lead to functional inactivation.The p53 tumor suppressor is not required for normal cellular proliferationbut rather functions as "guardian of the human genome" by integratingvarious signal transduction pathways that can sense cellular stress,including genotoxic and cytotoxic insults. The abrogation of p53 tumorsuppressor functions by high-risk HPV E6 leads to inhibition of the cyclindependent kinases that are critical for S-phase entry, allows the propagationof cells that have suffered genetic alterations and as such contributes togenomic instability. Considering the similarity of the genome structure andsequence between high-risk and low-risk HPVs, one can potulate thatlow-risk HPV E6 may play a similar role as high-risk HPV E6. The main purpose of the present study is to construct the recombinanteukaryotic expressive plasmid containing both HPV11E6 gene andenhanced green fluorescent protein (EGFP). Then, transfect it into humandermal fibroblast in vivo, and detect the difference of p53 expressionbetween the fibroblasts transfected and untransfected with the HPV11E6recombinant plasmid. There are three separate parts in this study: 1. Construction and identification of enhanced green fluorescentprotein reporter gene vector containing HPV11E6 gene According to the nucleotide sequence of HPV11E6, a pair ofoligonucleotides was designed as primers which contain nuncleotidesequence of EcoR I and BamH I restriction endonuclease at each endrespectively. The sequence encoding for HPV11E6 was amplified usingPCR technique. The PCR product was digested with EcoR I and BamH I,and cloned into the pIRES2-EGFP plasmid containing the reporter geneEGFP. The recombinant plasmid pIRES2-HPV11E6-EGFP was verifiedcorrectly by enzyme digestion and sequence analysis. Thus providing animportant and convenient tool to study intracellular function of HPV11E6. 2. Expression of the recombinant plasmid pIRES2-HPV11E6-EGFPin human dermal fibroblasts Human foreskin was cut to small pieces and digested with collagenaseand hyaluronidase. Then, collect the fibroblasts and cultured in DMEM at37℃ 5%CO2. Fibroblasts grown in six-well plates at a density of 1×105cells in 0.5 ml of DMEM per well were transfected with pIRES2-HPV11E6-EGFP plasmids DNA and pIRES2-EGFP empty vector DNA by means ofDOTAP in condition recommended by the manufacture (Roche). 12-48hafter tranfection, EGFP was observed by fluorescent microscope, mRNA ofHPV11E6 was detected by RT-PCR. Result suggests recombinant plasmidpIRES2-HPV11E6-EGFP successfully expressed in fibroblasts. 3. The primary function of HPV11E6 in human dermal fibroblasts The total proteins of human dermal fibroblasts and fibroblasts whichhave been transfected with pIRES2-HPV11E6-EGFP were isolated, andwere detected with p53 antibody by Western Blot. No difference...
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