Expression Of C Terminus Of HTERK And Its Effect On The Proliferation And Viability Of Cells

Background&ObjectiveTelomere exists in eukaryotes linear at the end of the chromosome, and its main function is to protect the biology chromosomal end against the enzymolysis by nucleic acid. It not only effectively prevents chromosomes from restructuring and degradation between each other, but also plays an important role in keeping stability and the structural integrity of the genom. In normal human cells, the telomere would be gradually shorten with cell division. When reduced to a certain extent, the chromosomal DNA will become unstable, and the cells enter the stage of exhaustion and the spiral unlock. At a result, the cells, though still survive, but will no longer divide and finally begin to natural aging or death.hTERT is an important part of the human telomerase. Studies have shown that, mRNA expression of hTERT has been highly detected in telomerase-positive primary tumors and cancer cell lines but not in telomerase-negative cells. Therefore, hTERT is one of the restriction enzymes that control telomerase activity. It is very important to the growth of tumor cells, then prevent the function of telomerase can allows telomeres normally shorten the length. And hTERT plays an role in the telomere and the tumor, which is not just simply activate telomerase, but the hTERT protein in the transport mechanism between the nucleoplasm and the nucleolus. The study confirmed that the gene sequences of the hTERT amino acids965-981st is a nucleolar localization signal. hTERT combined with telomere DNA3’overhang end, and the primer of telomere is located in the start of the telomerase RN A template, which is the key steps for telomeres to extend.The (TTAGGG)n is the binding site between hTERT and telomere, which is located in telomere DNA3’overhang end. Studies have shown that amino acid sequence the963-972nd, the993-1002nd, the1047-1056th, the1077-1086th and the1108-1117th of hTERT exist the protein binding activity to the formation of protein-DNA conjugates with (TTAGGG)n.In order to explore the positioning and binding situation of hTERT in human cells, we constructed recombinant plasmid containing amino acid sequence936-1132nd in the hTERT (named as hTERT C197, including nucleolar localization signal and five binding site).In order to preliminary study of hTERT C197role in human cancer cells and immortalized cells, the eukaryotic recombinant plasmid were transfected into Hela cells(cell of human uterine cancer) and293T cells(cell of human kidney), and then MTT assay was used to detect cell proliferation and cell viability. At last we analysis Hela and293T cell proliferation and viability through statistical methods.Methods1. Prokaryotic plasmid construction and expression of hTERT C1971.1Access of the exogenous gene fragmentsPCR primers were designed by primer design software and cloning sites district of prokaryotic expression vector. Both ends of the primers were introducted two specific restriction sites BamHI and Xhol. The cDNA of hTERT, as a template, was used to amplified exogenous gene sequences. The exogenous gene fragment was purified at the end.1.2Construction of recombinant plasmidProkaryotic expression vector pEGX-4T-1and exogenous gene fragments were double digested by BamHI and Xhol, and then the digestion product were ligated in thr T4ligase system. The ligation product was transformed into E. coli DH5a to construct the transformed bacteria. Extraction of prokaryotic recombinant plasmid and it was identificated by a series of experiment. 1.3Inducible expression of the target proteinThe prokaryotic recombinant plasmid was transformed into E. coli BL21(DE3) cells and it was identificated by a series of experiment. The recombinant protein was expressed in engineering bacteria by the IPTG induction. To determine the expression of recombinant hTERT C197, total protein was extracted and detected by western blot.2. Eukaryotic plasmid construction and expression of hTERT C1972.1Access of the exogenous gene fragmentsPCR primers were designed by primer design software and cloning sites district of eukaryotic expression vector. Both ends of the primers were the introducted specific restriction sites KpnI and XbaI. The cDNA of hTERT, as a template, was used to amplified exogenous gene sequences. The exogenous gene fragment was purified at the end.2.2Construction of recombinant plasmidEukaryotic expression vector pcDNA3.1-HisA and exogenous gene fragments were double digested by KpnI and XbaI, and then the digestion product were ligated in the T4ligase system. The ligation product was transformed into E. coli DH5a to construct the transformed bacteria. Extraction of eukaryotic recombinant plasmid and it was identificated by a series of experiment.2.3Inducible expression of the target proteinThe eukaryotic recombinant plasmid was transformed into Hela cell and293T cell. They are identificated by a series of experiment. The target protein is expressed in cells. We can extract the protein, and it can be identificated by Western blot at the end.3. Cell proliferation and cell activity of hTERT C1973.1Cell culture and transfectionHela cells and293T cells were resuscitated and subcultured by conventional cell culture.96-well culture plates inoculated with the cells.1×104cells/well of Hela cells and1.1×104/well of293T cells were inoculated plate respectively. The Hela cells and293T cells were transfered in seven96-well plates respectively, each plate used45holes. Adherent cells before experimental intervention.3.2Inhibition of cell proliferation and viability of cells.Both cells took a board to measure the OD value by MTT comparison method each day and7days of continuous measurement. Finally, we used the SPSS statistical software to statistical analysis of data in accordance with the experimental program. At last, we find the effect to the proliferation and viability of cell by hTERT C197.Result1. Exogenous gene fragment can be amplified by PCR.2. Prokaryotic recombinant plasmid can be constructed by the hTERT C197and prokaryotic vector pGEX-4T-1.3. The prokaryotic recombinant plasmid was transformed into host strain and the target protein is expressed by the IPTG induction.4. Eukaryotic recombinant plasmid can be constructed by the hTERT C197and prokaryotic vector pcDNA3.1-HisA.5. The eukaryotic recombinant plasmid was transformed into host cells. The recombinant protein is expressed in karyon of cells and it can be identificated by Western blot.6. Within seven days after the experiment intervention, according with the single factor analysis of variance, there is no significant difference in blank group and control group each day(P>0.05);There is significant difference in blank group and positive group each day(P<0.001);There is significant difference in control group and positive group each day(P<0.001).There is no effect on the proliferation and activity of Hela cell and293T cell by carrier plasmid. But there is significant influence in the proliferation and viability of Hela cell and293T cell by restructuring plasmid.7. hTERT C197can be decreased the proliferation and viability of Hela cell and293T cell. The maximal inhibition rate were both on the fifth day, there were33.07±0.229(%) and33.92±0.114(%)Conclusion 1. Prokaryotic recombinant plasmid pEGX-4T-1-hTERT C197was constructed successfully, and the target protein is expressed successfully in prokaryotic expression system.2. Eukaryotic recombinant plasmid pcDNA3.1-HisA-hTERT C197was constructed successfully, and the target protein is expressed successfully in eukaryotic expression system.3. hTERT C197can significantly inhibit the proliferation and viability of Hela cell and293T cell.