Construction and identification of PRDX3 eukaryotic expression vector / 军事医学

Objective To construct the eukaryotic expression vector of PRDX3 labeled with FLAG tag and to study its localization in human tongue cancer cell line SCC15.Methods PRDX3 gene was obtained from the breast library by PCR and cloned into PCDH vector to construct PCDH-FLAG-PRDX3.The plasmid was transiently transfected into 293T cells and the expression was detected by Western blot.Subcellular localization was detected by cellular immunofluorescence.Results The result of double digestion and sequencing showed that PCDH-FLAG-PRDX3 eukaryotic expression vector was constructed.The expression of FLAG-PRDX3 in human 293T cells was positively confirmed by Western blotting.In human tongue cancer cell line SCC15, the result of cellular immunofluorescence showed FLAG-PRDX3 was located in the cytoplasm rather than in the nucleus.Conclusion PRDX3 eukaryotic expression vector labeled with FLAG tag is constructed successfully, which is located in cytoplasm in human SCC15 cells.Construction and identification of PRDX3 could shed light on the function and mechanism of PRDX3 in tongue cancer.

Construction of eukaryotic expression vector of human telomerase RNA component and its function / 军事医学

Objective To construct the eukaryotic expression vector of human telomerase RNA component ( hTR) and study its biological function tentatively .Methods hTR Gene was obtained by PCR from cDNA template , which was reverse transcribed from 293T mRNA and cloned into pCDNA3.0 vector.The recombinant plasmid and empty vector were trans-fected into 293T cells, and hTR expression was identified by qRT-PCR.HepG2 cells that stably transfected with pCDNA3.0-hTR were constructed and identified by qRT-PCR.These cells were used to assess the interaction of hTR with human telomerase revese transcriptase ( hTERT ) and dyskerin .Telomerase activity was also detected in HepG 2 cells transfected with pCDNA3.0-hTR.Results pCDNA3.0-hTR eukaryotic expression vector was successfully constructed by double digestion identification .The inserted fragment was confirmed by sequencing .The expression of hTR in human 293T cells and HepG2 pCDNA3.0-hTR stable cell line was identified.In addition, qRT-PCR and Western blotting results showed that hTR could interact with hTERT and dyskerin , while hTR overexpression could not regulate the telomerase activity in HepG2 cells.Conclusion The eukaryotic expression vector of pCDNA 3.0-hTR is successfully constructed and expressed.This study will contribute to the further study of cancer therapy targeting hTR .