Expression and Antibody Preparation of sglt1 from Cyprinus CarpioL.
|Keywords||Cyprinus carpio L sodium/glucose cotransporter 1 (sglt1) prokaryotic expression ELISA Sglt1 antibody antibody titer Immunohistochemistry|
To systematically investigate the cloning, expression, antibody preparation and antibody titer of Na+ glucose cotransporter 1 (sglt1) in the intestine of Cyprinus carpio L., biological and immunological techniques including RT-PCR, SDS-PAGE, ELISA were used. The full-length of sglt1 was cloned. An antigenic determinant of 92 amino acids was expressed and this fragment of the epitope was purified. The antibody with high specificity for Sglt1was successfully obtained.1. Expression of the Sglt1 antigenic determinant. The Sglt1 antigenic determinant of Cyprinus carpio L. was successfully expressed. The full-length sglt1 from carp with an ORF of 1977 bp was cloned which could encode a protein of 658 amino acids. In this study, 92 amino acid antigenic determinant of sglt1 from carp was cloned into plasmid pET-32a (+) vector which had restriction sites for EcoRI and HindIII. The antigenic determinant of sglt1 in pET-32a (+) was then transformed into E.coli Rosetta (Engineered Bacteria of Sglt1). After recovering the expression products from the engineered bacteria induced by IPTG, a specific band of approximately 30 kDa was obtained on SDS-PAGE.2. Antibody preparation of Sglt1. New Zealand Rabbits were immunized ear margin veins with subcutaneous injection with the mixture of the purified fusion protion power and Freund’s Complete Adjuvant and boosted three times with the purified fusion protein power in Freund’s Incomplete Adjuvant four times and immune total duration for 38 days. The polyclonal antibody with high sensitivity (1:105) was obtained. The effect of antibody was verified by immunohistochemical. The results showed that the preparation of antibody can be applied to the carp Sglt1 expression locate studies.The Sglt1 polyclonal antibody of this study preparation as important research tools laid the groundwork for the Sglt1 expression of carp intestinal research systems and transshipment activity. At the same time, the Sglt1 polyclonal antibody also can be used Sglt1 transporters express position and express quantitative study of other fish.