Construction And Expression Of PE38KDEL Driven By HTERT Promoter On Human Pancreatic Cancer MiaPaCa2 Cells

Objective:Pancreatic cancer is one of the most common tumors and have a 5-years survival for all stages less than 5%.Most patients are diagnosed at advanced stage and therefore not candidate for surgical resection.The reasons for poor prognosis include: the difficulty of early diagnosis due to lack of specific early symptoms;the frequent occurrence of distant metastasis;the poor response to existing chemo-,radio-therapy, therefore it is necessary to establish gene therapy method.In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer.Telomerase is a ribonucleoprotein complex the function of which is to add telomeric repeats(TTAGGG)n to chromosomal ends,and it is known to play an important role in cellular immortalization.Telomerase is highly active in most tumor cells,yet not in normal cells.As such,it may have possible application in cancer gene therapy.Telomerase consists of two essential components,telomerase RNA template(hTR) and catalytic subunit(hTERT),hTERT is expressed only in cells and tissues positive for telomerase activity.Using the promoter to control the target gene to express only in tumor cells may be become one effective method of target gene theapy.Pseudomonas exotoxin(PE) A is a single chain peptide secreted by pseudomonas aeruginosa.Its molecular weight is about 660 000 and consisted of 613 amino acids.As biological immunotoxin,PE has prosperity in target gene therapy.PE38KDEL is a shorted PE which has a lighter weight and lower immunogenicity and highly toxicity.In this study,we constructed a plasmid containing PE38KDEL and enhance green fluorescent protein(EGFP)which only expressed in human telomerase reverse transcriptase(hTERT) positive cell line under the control of hTERT promoter.Methods:We replaced the promoter of pIERS2-EGFP with hTERT promoter from pGL-3 by polymerase chain reaction(PCR).Then we subcloned PE38KDEL into the multiple clone sites(MCS) of pIERS2-EGFP.The transfection of pIERS2-EGFP,phTERTp-PE38KEDL-IRES2-EGFP into MiaPaCa2 and Wi-38 was conducted by transfectmine 2000,and the effect was judged by observing the fluorescence expression of EGFP. Results:The correct construction of phTERTp-PE38KEDL-IRES2-EGFP was confirmed by bi-enzyme cut and sequence test.Transfected with pIERS2-EGFP,the EGPF can be observed in MiaPaCa2N HEK293 and Wi-38 cells by fluorescence microscope.The EGFP can be observed in MiaPaCa2 and HEK293,but can not be observed in Wi-38 with the transfection of phTERTp-PE38KEDL-IRES2-EGFP.Conclusion:The EGFP and PE38KDEL controlled by hTERT promoter can be expressed specifically in human pancreatic cancer cell lines MiaPaCa2 and.human embryonic cells(HEK 293) hTERT promoter may be used as an excellent regulation element in tumor targeting gene therapy.