| Telomerase maintains the telomeric ends of linear chromosomes and protects them from degradation and end-to-end fusion .The evidence has shown that telomerase is activated in more than 85% of cancer cells, but not in normal cells .Three components of human telomerase, human telomerase RNA component (hTR), human telomerase protein l(hTPl), and human telomerase reverse transcriptase (hTERT), have been identified. Among them, mRNA expression of hTERT has been highly detected in telomerase-positive primary tumors and cancer cell lines but not in telomerase-negative cells. Many evidents have demonstrated that peptides from hTERT are recognized by MHC class I ,which activated tumor specific CTL reaction that can kill tumor cells from different histological origin.The hTERT is a tumor-associated antigen expressed in the vast majority of human tumors and is presently one of the most promising target candidates for the immunogene therapy of cancer.Objective:To provide a sound basis for further researching cancer immunogene therapy ,recombinant plasmids of pcDNA3.1-hTERT and pGEX-4T-l- hTERT will be constructed ,and the expression of pGEX-4T-l-hTERT in BL21 will be observed.Methods and Results:1.Clone and sequence analysis of hTERT gene fragmentTo construct eukaryotic recombinant plasmid of human Telomerase reverse transcription(hTERT) ,total RNA was extracted from the tumor tissue according toQiagen manufacturer's instruction ,and the fragment of hTERT cDNA containing Hind III and BamH I sites was amplified by reverse transcription and polymerase chain reaction(RT-PCR).The amplified fragment was cloned into pGEM-T Easy vector . The recombinant plasmid was detected by Ampicillin antibiotic , blue-white screen and restriction endonucleases specific PCR,then was digested by Hind III and BamH I .The target fragment was subcloned into the Hind III and BamH I site of eukaryotic expression vector pcDNAS.l. The target fragment can be found when recombination transform into the competent cells E.coli JM109 ,and be identifiedd by restriction endonuclease and PCR.To construct prokaryotic recombinant plasmid of hTERT, the fragment of hTERT containing EcoR I site was amplified by RT-PCR and was cloned into pGEM-T Easy vector .The fragment was digested by EcoR I and subcloned into the EcoR I site of pGEX-4T-l expression vector. The constructed plasmid was denominated with pGEX-4T-l -hTERT and transformed into competent cells of E.coli BL21 .The pGEX-4T-l -hTERT recombinant plasmid was identified by specific PCR.The sequence of hTERT fragment was determined and analyzed by biological software Omiga2.0. Gene sequencing results : the hTERT fragment was 951bp long .It encoded the polypeptide of 317 amino acid residues,corresponding to calculated molecular masses of 37KDa .The homology in nucleotide acid of the hTERT fragment compared with the hTERT gene reported in GenBank was 98.73% , and the homology in amino acids was 99.68%.2.Expression of pGEX-4T-l -hTERT fusion proteinBL21 containing pGEX-4T-l -hTERT was inducted by l.OmM IPTG ,then was analyzed on SDS-PAGE of 12% gel. A 63KDa protein band of predicted molecular mass which presumably comprises of a GST and an hTERT was observed with samples induced by IPTG ,but not in the samples without IPTG induction. The level of expression peaked at 4h post-incubation. The portion of the fusion protein reached 28.4% of all the proteins by thin-layer gel optical scanning. The fusion protein was also analyzed by Western-blot. The results showed that the fusion proteion has the better antigenicity and could be recognized by hTERT polyclone antibody.Conclusions:l.The selected hTERT gene fragment is highly conserved genes ,and can be taken as candidate gene in tumor vaccine developing.2.The recombinant plasmids of pcDNA3.1-hTERT and pGEX-4T-l-hTERT were constructed successfully.3. Western blot analysis confirmed that 63KDa fusion proteion of pGEX-4T-l-hTERT could be specifically recognized by hTERT polyclone antibody.This result sugge... |
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