The Fusion Expression Of Human Na～+/Dicarboxylate Cotransporter1 And Its Role In Pathogenesis Of Nephroliathisis

The human Na+ldlcarboXylat6 cotransport6r medlat6s the transport of Krebs cyclelntermedIat6s, such as succInate, CItrat6 and a-oxogIut9rat6, across the pIasmamembrane of mammalIan cehs lt has been consldered to be an lmportantdetermInant of unnary CItrat6 concentratlon and be InvoIved ln energy utIIlzatlon asweIl as synthesls Of amlno acId and gIucocortIcost6roId RecentIy lt was report6dthat hNaDC1 mlght be a key f8Ctor In reguIatIng the llfdespan Of mammaIIan CItrat6ls an lmportant substrat6 fOr energy metaboIlsm and other blochemIcaI reaCtIons ItcheIat6s caICIUm ln the urlne and prevents the fOrmatIon of caIclum-contalnIngstOnes Theed re, lt Is valuabIe to elucldat6 the funCtlon of hNaDC1 and ltsSlgnlficance of pathophyslology In thIs study we firstIy predlcated the antIgenICIty ofamlno acld sequence of hNaDC1 and obtalned ltS bIochemIcaI param6ters bysoftware on-Ilne (httPthexpasypku edu cntol-blnlProtparam), then we seleCted thesequence of the deduced epltope fOr the fusIOn proteln The cDNA encodIng amlnoaCIds 159-215 of the hNaDC1 was produced by RT-PCR The PCR fr8gment was30punfied and digested with EcoR I and SaL I, then cloned into the EcoR I and SaL I sites of the expression vector (PGEX-5X-1) The identity of the recombinant plasmid was verified by sequencing The positive plasmid was transformed into E coli BL2I The clone harboring plasmid pGEX-hNaDCI was cultured and induced with IPTG The fusion protein of 36KD was produced at a level of 37% of the total cellular protein The fusion protein was punfied by GST Sepharose 4B affinity chromatography with yield of 25mg/mi of culture, and was used as an tmmunogen to inoculate rabbits The antibodies against the GST-hNaDCI fusion protein was raised Its specificity was identified by Western blot and immunohistochemtstic staining respectively The above results indicate that the antibody against GST-hNaDCI we made in this experiment could specifically recognize the fusion protein of 36KD and the hNaDCI located in the brush border membrane of renal proximal tubule, suggesting that the antibody could be used to study the biologic function and pathogenic role of this transporter...