Objective To evaluate the expression of telomerase transcriptional elements-interacting factor （TEIF） in human testis under different status and its relation with human telomerase reverse transcriptase （hTERT） exp...Objective To evaluate the expression of telomerase transcriptional elements-interacting factor （TEIF） in human testis under different status and its relation with human telomerase reverse transcriptase （hTERT） expression. Methods Specific antisera against TEIF were generated by immunization of rabbits with purified recombinated partial TEIF. Samples were assigned to three groups according to their pathological types, including 16 normal testes, 8 atrophic testes, and 6 testicular seminomas. They were subjected to immunohistochemical staining of TEIF and hTERT. Results from both TEIF and hTERT were analyzed semi-quantitatively and compared. The expressions of TEIF and hTERT were detected in all samples of normal, atrophic testes, and seminomas. No differences of TEIF expressions among these three groups were observed （P 〉 0.05）. On the contrary, the expressions of hTERT were significantly lower in atrophic testes compared with those of normal testes and seminomas （both P 〈 0.05）. Nevertheless, co-expressions of TEIF with hTERT were revealed to be in normal and malignant cases （P 〈 0.05） but not in atrophic testes, which generally presented TEIF expression. The cellular distributions of both proteins were similar and mainly in spermatocytes and some Sertoli cells, while were all negative in the interstitial cells and other stromal cells. Conclusion The uniform expressions of TEIF in all these specimens suggest that it may be a marker of testis and its related diseases. The strong expression of hTERT in normal testes and testicular seminomas comparing with the low expression in atrophic testes may suggest a role for telomerase in maintaining proliferation of germ cells.展开更多
To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for futher study Methods. The gene for encoding hTRT catalytic domain was cloned b...To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for futher study Methods. The gene for encoding hTRT catalytic domain was cloned based on RT PCR amplification from HeLa cells and sequenced The cloned hTRTcDNA was in frame inserted into His tag fusion expression vector pEK318 The His tag hTRT fusion proteins were purified by Ni NTA chromatography and stained by western blotting Results. An approximately 620bp fragment was generated and cloned into pBluescript SK+between SalI and BamHI sites DNA sequencing showed the isolated fragment was consistent to those reported SDS PAGE present that a 17kDa protein was expressed stably in E coli JM109 harboring pEKTRT344 containing 6×His tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6×His tag and hTRT 243aa was only detectable as 27 kDa band in western blotting Both of fusion proteins were purified by Ni NTA chromatography and showed single band(>95% purifity) in Coomassie Brilliant staining Western blotting confirmed that two proteins could be recognized by the Ni NTA AP conjugate Conclusions. The hTRT catalytic domain was highly conserved The expressed hTRT protein contained recognizable His tag, telomerase specific and strong antigenic epitops, which may be convenient for further investigation展开更多
基金Supported by N ationalN aturalScienceFoundation ofC hina (30170364 )and a grantfrom the CenterofH um an G enetic D isease,Peking Univ-ersit.y
文摘Objective To evaluate the expression of telomerase transcriptional elements-interacting factor （TEIF） in human testis under different status and its relation with human telomerase reverse transcriptase （hTERT） expression. Methods Specific antisera against TEIF were generated by immunization of rabbits with purified recombinated partial TEIF. Samples were assigned to three groups according to their pathological types, including 16 normal testes, 8 atrophic testes, and 6 testicular seminomas. They were subjected to immunohistochemical staining of TEIF and hTERT. Results from both TEIF and hTERT were analyzed semi-quantitatively and compared. The expressions of TEIF and hTERT were detected in all samples of normal, atrophic testes, and seminomas. No differences of TEIF expressions among these three groups were observed （P 〉 0.05）. On the contrary, the expressions of hTERT were significantly lower in atrophic testes compared with those of normal testes and seminomas （both P 〈 0.05）. Nevertheless, co-expressions of TEIF with hTERT were revealed to be in normal and malignant cases （P 〈 0.05） but not in atrophic testes, which generally presented TEIF expression. The cellular distributions of both proteins were similar and mainly in spermatocytes and some Sertoli cells, while were all negative in the interstitial cells and other stromal cells. Conclusion The uniform expressions of TEIF in all these specimens suggest that it may be a marker of testis and its related diseases. The strong expression of hTERT in normal testes and testicular seminomas comparing with the low expression in atrophic testes may suggest a role for telomerase in maintaining proliferation of germ cells.
文摘To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for futher study Methods. The gene for encoding hTRT catalytic domain was cloned based on RT PCR amplification from HeLa cells and sequenced The cloned hTRTcDNA was in frame inserted into His tag fusion expression vector pEK318 The His tag hTRT fusion proteins were purified by Ni NTA chromatography and stained by western blotting Results. An approximately 620bp fragment was generated and cloned into pBluescript SK+between SalI and BamHI sites DNA sequencing showed the isolated fragment was consistent to those reported SDS PAGE present that a 17kDa protein was expressed stably in E coli JM109 harboring pEKTRT344 containing 6×His tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6×His tag and hTRT 243aa was only detectable as 27 kDa band in western blotting Both of fusion proteins were purified by Ni NTA chromatography and showed single band(>95% purifity) in Coomassie Brilliant staining Western blotting confirmed that two proteins could be recognized by the Ni NTA AP conjugate Conclusions. The hTRT catalytic domain was highly conserved The expressed hTRT protein contained recognizable His tag, telomerase specific and strong antigenic epitops, which may be convenient for further investigation
