Cloning And Expression Of CDNA For Human Telomerase Catalytic Subunit HTERT Active Site

Abstract:Telomerase is ribonucleoprotein complex in eukaryocyte, which is composed of telomerase reverse transcriptase(TERT) and telomerase RNA. Telomerase is a special DNA polymerase that can extend the terminal of DNA and maitain the length of telomere. Its RNA subunit provides template for telomere synthesis. TERT have reverse transcriptase activity. Telomerase activity was not detected in most somatic cell and primary cell, but was shown in most tumors. Telomerase is indispensable for telomere extension and maintenance of cell division. Telomere locates on the terminal of DNA containing repeat DNA sequence. The repeat DNA sequences of various organisms were different. Te]omere can maintain chromosome stabilization. Telomere plays important role in maintaining nucleus 3D structure and insuring chromosome duplication. Telomerase catalytic subunit active site contains motifl, 2. A, B, C, D, which cover 1 764bp of DNA. Total RNA was extrated from the human tumor cell which possess strong telomerase activity using Taq plus DNA polymerase. There is a special band about 2000bp. The fragment was connected with clone vector pMDI 8-T. Then the pMDI 8-T-hTERT vector was transformed into competent cell of E.coli JMIO9. The plasmid was extracted from the positive clone and digested by FeaR I and HindIII in order to certify that the 2Kb fragment was successfully obtained. Sub~equentIy, the fragment was digested with BctmH I to identify the direction of fragment, meanwhile, the nesting PCR was used in order to identify the fragment correctness. The positive plasmid was sequenced. The result proved that the fragment is correct. After the gene connected with pMDI8-T vector was digest by EcoR I and HindIII, the fragmen was retrieved using DNA fragment retrieving kit, meantime. the eukaryocyte expressing vector pCR3. I was digested by EcoR I and Hind III and retrieved. The fragment and vector were ligated by ligase and then transformed into competent cell of E.coli JMIO9. After 12-18 hours of culture, several clones appear on the plate. Some positive clones were selected to extract their ptasmid. The~e plasmids were digested by EcoR I and JfindIII and indentified by PCR. The result showed that the hTERT have successfully ligated with eukaryocyte expressing vector pCR3. I. The pCR3. I vector has CMB promoter that is highly efficient for expression of target protein. The positive plasrnid was extracted using plasmid quick extraction kit. Liposorne was used as intermediate to transfect the pCR3.l-hTERT plasmid into DK cell. We extracted total RNA from the DK cell which was transformed with positive plasmid. mRNA was identified by RT-PCR. The result show that hTERT cDNA active site was successfully expressed in eLlkaryocyte.