Expression Of HTERT Protein And Screening Of Mimic Epitopes

The extreme ends of linear eukaryotic chromosomes contain specialized DNA-protein structures called telomere which cap the ends of chromosomes, preventing chromosomal degradation and fusion, and allowing for continuous cell proliferation. Telomerase is a unique RNA-dependent DNA polymerase with specialized reverse transcriptase characterization that uses a short templating sequence which contained within its RNA subunit to direct synthesis of telomeric repeats by the catalytic protein component, TERT (telomerase reverse transcriptase).Human telomerase reverse transcriptase , hTERT , has been identified as a catalytic subunit required for telomere elongation , it is the rate-limiting factor for telomerase activity both biologically and enzymatically . The correlation between positive for high telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase. Most normal human somatic cells do not have detectable telomerase activity and lack expression of hTERT, whereas most immortalized cells and approximately 85% human cancers demonstrate up-regulated telomerase activity and express hTERT. Recent studies reveal that mRNA of the human telomerase catalytic subunit protein, hTERT, has been shown to correlate strongly with telomerase activity. During human tumorigenesis, telomerase becomes reactivated by transcriptional up-regulation of hTERT . One critical function served by hTERT reactivation during cancer progression is to avert the adverse consequences of telomere shortening and loss of chromosomal capping function.Taken together , these findings indicate that the expression of hTERT as the rate-limiting step in telomerase activity and bring the study of hTERT gene expression to the forefront of telomerase regulation research, making ita valuable target in cancer diagnosis. Therefore, Further understanding of the mechanisms involved in hTERT regulation is necessary of great importance and hTERT become a promising target for new strategies of drug development for anti-cancer as well as the treatment of other age- related diseases.Phage-displayed peptide libraries emerge as a proven method for the exploring of novel biologically active molecules and offer an attractive approach for the identification of discontinuous and linear and even nonproteinaceous peptide ligands for various targets. Most peptides selected by phage display bind at sites that coincide with natural ligand binding sites, and consequently act as agonists or antagonists of protein-protein interaction. A short, active peptide could then be used as a leading compound for the rational design of small, non-peptide orally available therapeutic molecules.The study can be divided into following three parts: Part 1 Expression and purification of telomerase reverse transcriptaseClone 1.3kb foreign DNA insert of telomerase catalytic subunit gene which contains all 8 motifs and was amplified by PCR into expression vector of pET-32a. The recombinant plasmid was induced by IPTG for 4h and produced 59KD recombinant protein which appeared in the form of inclusion body. This inclusion body was dissolved in 8mol/L urea and purified by affinity chromatograph using Ni-NTA resin under denaturing conditions. Purified hTERT protein was identified by SDS-PAGE and Western-Blot. Results revealed hTERT functional region recombinant protein was expressed and purified correctly.Part 2 Prepariation of mouse multi-clonal antibody target hTERTImmuned normal mice using purified hTERT protein by standard method to obtain polyclonal anti-hTERT antibody. Dot-ELISA and ELISA identified that polyclonal antibody responsed specifically to recombinant hTERT protein in a concertation correlated manner, the titering of this antibody serum achieved 1:64000. After purification of this polyclonalantibody through SPG column according the protocol and the antibody with such characteristics can be used as target in the three-round screening of phage display peptide library.Part 3 Screening and identification of hTERT e...