Construction and expression of the eukaryotic vector for the gene encoding the urea channel protein(Urel)of Helicobacter pylori and investigation on its antigenicity
Abstract：The recombinant eukaryotic vector for gene encoding the urea channel protein(Urel)of Helicobacter pylori was constructed and expressed in COS-7 cells for the sake of providing a foundation to develop the nucleic acid vaccine.Under this subject,the eukaryotic expression plasmid pET32a(+)/UreI was used as template and the target gene fragment encoding the UreI protein was amplified from this plasmid,digested with Hind Ⅲ and KpnⅠ.In the same way,plasmid pEGFP-N1 was also digested with these two endonucleases simultaneously.Then,the objective gene and plasmid pEGFP-N1 were purified with gel kit and connected by T4 ligase at 4∶1 molar rate.The recombinant vector pEGFP-N1/UraI was used to select,transform and meantime express in COS-7 cells.Meanwhile,the expression of this eukaryotic vector in Cos-7 cells was investigated using fluorescence microscopy and Western blotting.As demonstrated by restriction endonuclease analysis,and sequencing,the size of the inserted target gene fragment was found to be 585 bps.And this gene fragment was proved to be the gene encoding the UraI protein.As compared with the gene of H.pylori AM417609 reported by GenBank,the cloned ureI gene sequence was completely coincidental.The expression of recombinant vector pEGFP-N1/Urel in COS-7 could be detected by means of fluorescence microscopy and recognized by the anti-H.pylori antibody of patients infected with this organism,suggesting that this protein had good biological activities.It is evident that the recombinant eukaryotic vector p EGFP/UreI has been constructed and successively expressed in COS-7 cells.
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