Tert Promoter Cloning, Activity Identification In Mice And Its Regulation Of Gene Expression

Tumor is a frequently-occurring diseases which threats to human health seriously. Reports indicate that with its mortality rates only slightly lower than that of cardiovascular and cerebrovascular diseases, tumor is the second major threat to human life. Most traditional anti-tumor drugs are cytotoxic ones, and these drugs are often non-selective for normal tissues. They have a greater cytotoxicity on normal tissues, and are easy to produce drug resistance. Thus the development of high efficiency, low toxicity, specific new anti-tumor cancer drugs become more and more important. In recent years, during the development of molecular biology, molecular genetics, immunology and other related disciplines, the technology of cancer gene therapy developed rapidly, the level of some gene-targeted antitumor drugs were being developed. At this point, tumor gene treatment with its unique advantages has become research hot spots.Telomerase, which is composed by RNA, protein components and telomerase reverse transcriptase (TERT), is a ribonucleoprotein (RNP) that can catalyze the extension of the telomere, and telomerase reverse transcriptase (TERT) is the most inportant factor of telomerase activity. Telomerase did not express in most normal cells and showed higher expression in most tumor cells, therefore, the use of promoter regulation of telomerase gene expression, often used as a strategy of tumor-targeted gene therapy. At present, the studies of human TERT promoter transcriptional activity region was almost clear, but mouse TERT (mTERT) promoter transcriptional activity in the region is not very clear.Straphylococcal enterotoxin A is a powerful immunostimulant, which without antigen-presenting cell processing can be combined in the form of complete protein to the lateral of MHC-Ⅱmolecules, and at the same time produce a large amount of cytokines such as TNF-α, IL-2, IFN-γ, etc., and so as to result in anti-tumor effect. Superantigen-activation of lymphocytes needs cooperation of TCR-and CD28-mediated activating signals. CD80 is one of the co-stimulatory molecules binding CD28, which binds CD28 on the surface of T cells to provide second signal for efficient activation of T cells.Therefore, this study cloned different length of mTERT promoter fragment to study the activity in a variety of tumor cells and normal cells, and to clear the core of mTERT promoter region; used SEA and CD80 gene as a therapeutic gene, and the mTERT core promoter as a regulatory elements, to built a eukaryotic expression vector, then observed the expression in the tumor cells and normal cells, and provided the experimental material and theoretical basis for the tumor-targeted gene therapy.1. Cloning of mTERT promoter and study on the activity in different tumor cellsObjective:To clear the mouse TERT core promoter and use it as a tumor target gene regulatory elements in gene therapy. Methods:Extracted genomic DNA of Hepal-6 cells, and used it as a template, using PCR amplification methods to gain promoter fragment of mTERT1 (1086bp) named mTERT1, after Nco I and Xho I digestion cloned PCR products of Gel into pGL3-Basic vector to construct recombinant plasmid pGL3-mTERT1. Then used the plasmid pGL3-mTERT1 as a template, amplified different length mTERT promoter fragment by PCR, using the same method and cloned into pGL3-Basic vector, and they were named pGL3-mTERT2 to 7. After that transiently co-transfected plasmid pGL3-mTERTl to 7 and pRL-CMV into tumor cells Hepal-6, B16, CT26 and normal cells NIH3T3 by LipofectamineTM 2000, double luciferase method to detective the activity of mTERT promoter fragment in the cells. Used the activity of pGL3-Control as 1, the fluorescent activity of recombinant plasmid pGL3-mTERT tangled with the activity of pGL3-Control ratio was the relative transcriptional activity of mTERT promoter fragments with different length in the cells. Finally, by comparing the transcriptional activity of various fragments of mouse TERT promoter to identify the core areas, as well as to be a target gene regulatory elements in cancer gene therapy. Results:The cloning of different length mTERT promoter fragments identified by restriction enzyme digestion and sequencing with the sequence published in GenBank fully consistent; Hepal-6, B16 and CT26 cells have much higher transcriptional activity than the NIH3T3 cells, mTERTl-5 relative transcription activity is much higher than mTERT6 and mTERT7, in which the relative transcriptional activity mTERT5 higher, as mTERT6 and mTERT7 are almost zero. The relative transcriptional activity are different in different cells. Conclusion:This study shows that mTERT5 is core promoter region in different length of mTERT promoter fragment, and it can be the gene regulatory element in tumor target gene therapy next step.2. The identification of mTERT promoter regulation SEA, CD80 gene co-expressionObjective:To construct tumor-targeted gene regulation SEA/CD80 co-expression vector, and identified the expression of SEA and CD80 in the tumor cells. Methods:Insert the fragment that recovered from gal to the vector of pGL3-mTERT5 which had already been built. After digestion by the use of liposomes, transfect them (SEA+IRES+CD80) into Hepal-6, B16, CT26 and NIH3T3 cells by LipofectamineTM 2000. Then extracted RNA, used RT-PCR to identify the expression of SEA and CD80 in the cells, use anti-SEA antibodies(FITC tag) and anti-mouse CD80 antibody(PE tag) for immunofluorescent staining after that, last observed the co-expression of them. Results:The vector is right according to the identification of restriction enzyme digestion; RT-PCR results showed that, in the Hepal-6, B16 and CT26 cells transfected with pGL3-mTERT5-BIS plasmid, SEA and CD80 mRNA have a high level expression, but in the NIH3T3 cells transfected with pGL3-mTERT5-BIS plasmid, SEA and CD80 mRNA have a very weak expression; the result of immunofluorescent staining use fluorescence microscopy showed:SEA and CD80 in mice Hepal-6, B16 and CT26 cell membrane co-expression, but not in NIH3T3 cells. Conclusion:In this study we had constructed a SEA/CD80 co-expression vector of tumor-targeted gene regulation; SEA/CD80 can co-expression in Hepal-6, B16 and CT26 cell membrane, but not in NIH3T3 cell membrane. This study had provided theoretical basis and experimental materials for the next step of tumor-targeted gene therapy. This study confirmed that the core promoter region of mTERT is mTERT5, based on this conclusion we constructed a co-expression vector of SEA and CD80, pGL3-mTERT5-BIS, and that SEA and CD80 were co-expressioned in Hepal-6, B16 and CT26 cells, but not in NIH3T3 cells. This study had provided theoretical basis and experimental materials for the next step of tumor-targeted gene therapy.