Expression of sglt1 gene in Cyprinus carpio and preparation of its polyclonal antibody

Abstract

The high-affinity Na+/glucose contransporter Sglt1 is one of the important members of the sodium:solute symporter family(SSF),belonging to the homologous family 5(SLC5).The Sglt1 plays an important role in accumulating glucoses from intestinal or kidney epithelial cells against an adverse concentration gradient and maintaining the adjustment of metabolism.So far,few studies on Na+/glucose cotransporter have been reported in the freshwater fishes.This research would develop special antibodies of Sglt1,which could supply the foundation for the research of glucose metabolism in Cyprinus carpio intestines on molecular level.To study the molecular mechanism of glucose metabolism in freshwater fishes,the full-length cDNA of sglt1 in C.carpio with an ORF of 1 977 bp was cloned and the antigenicity of Sglt1 was first predicted.92(544-637)amino acids with strong antigenicity and immunogenicity were selected as target section of sglt1 in C.carpio and were cloned into plasmid pET-32a(+)vector which had restriction sites for EcoRⅠ and HindⅢ.The rcombinant plasmid named pET-32a(+)-sglt1-P was transformed into E.coli Rosetta.The Sglt1-P fusion protein of approximately 30 ku was highly expressed in E.coli Rosetta after being induced with IPTG.The purified fusion protein was used as antigen to immunize New Zealand Rabbits with ear margin veins by subcutaneous injection with power.The results of ELISA showed that the titer of the antiserum was about 1∶ 105.The Sglt1-P polyclonal antibody was used to determine the expression of Sglt1 protein in C.carpio intestines through immunohistochemical method.The result revealed that the antibody performed high affinity and specificity and could be applied to study the expression and locating of Sglt1 in the C.carpio.The Sglt1-P polyclonal antibody also could serve as an important research tool to study Sglt1 expression and transshipment activity of C.carpio.Meanwhile,the Sglt1-P polyclonal antibody could also be used to exploratively study the expression location and quantity of the Sglt1 transporters in other fishes.