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Construction Of Eukaryote Polycistronic Expression Vector And Expression In Hela

Posted on:2008-08-12Degree:MasterType:Thesis
Country:ChinaCandidate:S P MiaoFull Text:PDF
GTID:2120360215991482Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Endostatin cDNA was obtained by RT-PCR from the human liver total RNA and then inserted into pEF1-4SX to form a interim vector pEF1-es. Then the fragment of endostatin linking with IRES was cut and ligated into eukaryotic expression vector pIRES2-EGFP-FHIT to construct the tricistronic eukaryotic expression vector pES-IRES-FHIT-IRES-EGFP which contains endostatin, Fragile histidine triad(FHIT) and enhanced green fluorescent protein (EGFP). This recombinant vector was transfected into Hela cells with LipofectamineTM 2000 to check for their expression. 24h later, the green fluorescence emitting from the transiently tranfected cells was observed under fluorescence microscope. Total RNA was extracted from those stably transfected Hela cells selected with G418, and then was checked by RT-PCR with primer combinations of P1 vs P2, P3 vs P4, P5 vs P6 and P1 vs P4, respectively. The results of RT-PCR indicated that the moncistrons of endostatin, fragile histidine triad(FHIT) and enhanced green fluorescent protein (EGFP) as well as the tricistron of these three genes all had been transcribed successfully in the transfected Hela cells. Southern blotting of the total RNA also confirmed this conclusion. Further more, the results of immunocytochemistry with rabbit anti-FHIT antibody and rabbit anti-endostatin antibody showed that FHIT and endostatin had been translated in the transfected Hela cells. These results provided some basic information for further research on multigene therapy strategies in tumor treatment.
Keywords/Search Tags:polysistron, IRES, transfection, southern blot, northern blot
PDF Full Text Request
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