| EBNAl, a unique viral protein found in the four forms of latent infection by Epstein-Barr virus (EBV), provides a distinct episome maintenance function by binding to oriP, the latent origin of DNA repliction. In this study, based on the interaction of EBNAl with oriP, the element of oriP and SV40 promoter was engineered into the upstream of the El region of adenoviral genome to generate recombinant adenoviruses. Taking EBNAl for the targeted gene, the recombinant adenoviral replication and oncolysis can be restricted in the EBNAl positive tumor cells, and ultimately kill the EBV-associated tumor cells, which provide the new therapy approach to EBV-associated tumor. The research has been divided into three parts:1. Construction of EBNAl positive epithelial cell strain.Plasmid pcDNA3/EBNAl was derived from pcDNA3 by inserting the l,200bp gene fragment which encodes EBNAl and was transfected into human nasopharyneal cancer CNE2 cell and human cervical cancer Hela cell by Lipofectamine Reagent . Clones of cells were screened by G418 and identified by PCR, RT-PCR and Western blot assays. These results indicated that the two transfected EBNAl gene cell lines could express the EBNAl consistently and to be good tools for research of EBV- associated tumor.2. Enhancement of Epstein-Barr virus latent origin of replication on transcription.OriP is a 1.7-kb region of the Epstein-Barr virus chromosome and functionally maintains replication and stable presence of the virus-based plasmid in human cells that contain Epstein-Barr virus nuclear antigen 1 (EBNAl). This study was designed to screen the best fragment from oriP which enhances the transcription in EBNAl positive cells. A series of CAT plasmids containing different fragments of oriP were constructed and transiently transfected into EBNAl-positive B95-8 cells by electroporation. Cells were harvested after 48h culture and chloramphenicol acetyl transferase(CAT) levels were assayed. These results demonstrated that the FR2 fragment from 7,394bp to 8,04Ibp was the best enhancer on transcription and the relative activity of CAT reporter in the plasmid containing FRa had the most significant increase which was 4.8-fold the plasmid containing oriP sequences in B95-8 cells.3. The selective killing effect on EBNA1 positive tumor cells of recombinant adenovirus.The element FR and SV40 promoter was engineered into upstream of the El region, which is essential for viral replication of adenoviral 5, so that two kinds of recombinant adenoviruses ( one having E3 region named Q2E3 and the other lacking E3 region named Q2AE3) were generated. EBNA1 negative cells served as the control, viral replication and the cytopathic effect of QE3, QaAES and wild-type Adenovirus were evaluated by MTT assays in lymphoma cell lines and CNE2/EBNA1. The assays attained such results as follows : (1) . Both the recombinant adenoviruses Q2E3 and Q2AE3 could provide significant effect on killing EBNA1 positive cell lines. Cells viabilities of the EBNA1 positive cells were 23-61 and the EBNA1 negative cells were 90-98 at a MO of 1,000. (2). There was no remarkable difference between Q2E3 and Q2AE3 in killing effect on EBNA1 positive cell lines (>0.05). (3). Compared with QE3 and QAE3, the WAd had no specifically killing effect on the EBNA1 positive cell lines. Their survival rate were 19-50 infected with 1,000 MOI. (4). QE3 had strong killing effect on CNE/EBNA, The survival rate of CNE/EBNA1 was 21%, ofCNE2 was 37% , and of the negative control MRC-5 was 94% at a MOI of 1 (P<0.05). The results proved that the recombinant adenovirus Q2E3 and Q2AE3 had the function of targeted therapy on the EBNA1 positive tumor.
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