[Cloning, expression of a segment of hTERT gene and detection of telomerase and hTERT by anti-hTERT polyclonal antibody].
Telomerase is an important biomarker in cancer cells. It is active in germline cells, most of cancer tissues and cell lines, but not in most somatic tissues. Telomerase is composed of two components, and while hTER is present in normal and tumor cells, expression of hTERT appears to be highly regulated and correlates with telomerase activity. In order to detect the telomerase enzyme and hTERT protein, anti-hTERT polyclonal antibodies were produced in this study. A segment of hTERT cDNA was amplified by RT-PCR and cloned into the multi-cloning site of the GST gene fusion vector pGEX-5X-3. After the recombinant plasmid was expressed in E. coli BL21, the fusion protein was purified for immunization. Extracts from several cultured cells were analyzed by Western blot, and the results indicated that telomerase enzyme and hTERT protein could be specifically detected by this anti-hTERT antibod'. Thus, a simple and effective method was primarily established for the immunodetection of telomerase enzyme and hTERT protein.
Saleh
S A
SA
Institute of Genetics, State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433.
Sun
J L
JL
Chen
Z
Z
Huang
J S
JS
Shen
X R
XR
Zhang
B S
BS
Ren
D M
DM
D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D019098:Telomerase / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D019098:Telomerase