Design And Construction The Gene Elements Of The Human Papillomavirus 11 Intact Genome Transgenic Mouse

Human papillomavirus (HPV11) is the most major pathogen of the Condyloma acuminatum (CA). At present, there is no way to radical cure CA. The pathopoiesis of this disease are unclear. Therefore, an ideal animal pathology model of CA is very significant for the research of pathogenesis in vivo. And it's also important for the evaluation of the therapeutic methods in preclinical. The main content of this research is to design the exogenous gene of the HPV11 transgenic mouse model and construction it in the plasmid. A prerequisite of the expression of HPV11 pathogenic protein is the state of the HPVllgenome are free from the chromosome of the host. Additionally, this virus invades the epithelial exclusively. Accordingly, the transgene should contain the HPV11 complete genome. Inaddition, it should contain the gene elements which have the function of liberate the HPV11 genome from the host chromosome in the epithelial cell of the transgenic mouse.Based on the pathopoiesis characteristics of the HPV11 and the controlled expression mature technique Cre/LoxP, the genome sequence which will be used to introduce into mouse were designed. The major idea is to insert the complete HPV11 genome between the LoxP sequence in the sequence of 3' -5' .The eukaryote promoter and the EGFP are beside each LoxP separately. When the exogenous Cre primed by epithelium promoter, the HPV11, which is in the middle of LoxP will be liberated. Consequently, the uncontrolled condition of the HPV11 in the human will be reproduct in the mouse. At the same time, the residue of the LoxP will connect the promoter with the EGFP. The fluorescence is going to appear.The first part of the experiment is the plasmid construction. Our laboratory conserves the complete HPV11 genome in the plasmid pBR322/HPV11. The plasmid pQE-Trisystem which is qualified to construct the function sequence beside the HPV11 has been procured also. The procedure of plasmid construction is divided into three small parts. Part one: the pBR322/HPV11l and pQE-Trisystem was digested with the BamHâ… . The pQE-Trisystem was then dephosphorylated. After the ligation procedure, the product of the ligation was transformed into the DH5a. The positive clone was then screening by colony PCR. The product named Plasmid A. Part two: first to PCR the Loxp-EGFP-PolyA sequence from the pIRE2-EGFP. The restriction enzyme sites was been added meanwhile. Then the products of PCR and the plasmid A was been digest with the same restriction enzyme., and then ligated. The product named Plasmid B. Part three: first to amplificate the LoxP-7931-7072 from the pBR322/HPV11. The restriction enzyme sites was been added meanwhile. Then the product of PCR and the plasmid B was digest by the same restriction enzyme, and then ligated. After transforming into the DH5a, the positive clone was then screening by colony PCR. The output is the final product which named plasmid C.The second part of the experiment is to test the function of this sequence in vitro. First of all, the keratinize cell (KC) was separated from the acrobystia of children. Then the Plasmid C and the pIC-Cre which can express the Cre was cotransfected into the KC successfully in the second generation. The control group was adopted as well. The fluorescence was observed and the RT-PCR was done to detect the expression of HPV11 E6 gene in transcriptional levelResult: 1.The agarose gel electrophoresis and sequencing was carried out to verificated the construction of plasmid C consistent with the expectation production.2. The fluorescence was been observed in the experimental group when transfected into KC.3. The E6 was been detected by RT-PCR in experimental group4. The times of passaged generations of experimental group are three times of the control group.Conclusion: The experiment in vitro manifests that the plasmid C reproduced the pathopoiesis feature of the Wt. Moreover the HPV11 can be liberated by Cre/LoxP intra-celluar. The fluorescin can express in effect. Therefore, the geneome in the plasmid C have the potential of producing the HPV11 transgenic pathology mouse model.