The Expression Of HTERT In Prokaryotic System And The Preparation Of Antibody Against HTERT

Background : Telomerase, a Ribonucleoprotein enzyme that can extend telomere of eukaryotic chromosome, consists of a catalytic subunit telomerase reverse transcriptase (TERT) and a telomere RNA subunit. About 85% of human tumors 'show positive in telomerase activity. Telomerase activity is very low or absent in normal cells, whereas it is up-regulated in immortalized cells.Objective : To prepare specific polyclonal antibodies (PAb) against human telomerase reverse transcriptase ( hTERT) and explore its application in the diagnosis and researches of human cancer.Methods: (1) The specific motif T of hTERT and the C region of hTERT were inserted in a prokaryotic expression vector, the recombinant protein were expressed and purified; (2) Polyclonal antibodies (PAbs) were prepared from rabbits immunized with the recombinant protein; (3) Immuno- histochemistry assay and WesternBlotting were performed to analyze the titer of Pab and detect the telomerase activity in human tumor cells.Results: (1) The titer of PAbs detected by Western Blot is from 1 :10000 to 1:20000 ;(2) Human tumor tissues were stained positive by Immunohistochemistry with the post-immunization sera compared to the pre- immunization sera; (3) A band of approx. 60-kDa was detected in extracts of 293 cells with the post-immunization sera by Western blotting..Conclusion: We have prepared high titer PAbs against hTERT.These Pabs may have potential application in detecting telomeraseactivities of human cancer.