Expression Of Intestinal Peptide Transporter PepT1from Cyprinus Carpio L.

As the main products of proteins digestion, small peptides play an important role in the digestion, absorption and metabolism of the amino acids. Peptide transporter1(PepTl) is investigated most widely, which major transport di-and tri-peptides. Employing Real time-PCR, SDS-PAGE protein electrophoresis and other molecular biology and biochemistry techniques, we investigated PepTl in the intestine of Cyprinus carpio L. We cloned the full length of PepT1cDNA. The immunogenicity gene fragment was expressed the and PepTl antiserum was prepared. Using ELISA and immunohistochemistry techniques, we analyzed PepTl expression characteristics in different organizations. At the same time, using Real time-PCR technique, we discussed the expression characteristics of PepT1gene in different tissues and how the oligopeptides effected the expression of PepTl gene. The results as follows:1. Recombinant expression of the immunogenic peptide. In this experiment, we took the carp intestine as the material and cloned the full-length of PepT1gene’s open reading frame. We selected pET-32a (+) as expression vector, introducing the BamHI and EcoR I restriction endonuclease sites. Immunogenic PepT1fragment (198bp) encoding66amino acids was connected to the pET-32a (+) Vector. The recombinant vector was transformed into E. coli Rosetta, and with the induction of1‰IPTG, the gene engineering bacteria expressed immunogenic PepTl fragment successfully. SDS-PAGE electrophoresis analysis showed:The molecular weight of target polypeptide was about28kDa.2. Preparation of PepTl antibody. The purified fusion protein was emulsified with complete Freund’s adjuvant and incomplete Freund’s adjuvant. Then we used the antigen to immune the New Zealand Rabbits, which were immuned through hypodermic and ear margin veins injection four times to get antibody. The titer of the antiserum was about4×105through ELISA.3. Tissue distribution of PepTl. Expression and localization of PepTl protein in the gut, kidneys, spleen, liver and spleen were studied through immunohistochemical methods. The results showed, PepTl protein showed positive staining in the foregut, midgut and hindgut; and positive staining could also be observed in the spleen, liver and kidney. Using Real time-PCR, PepTl mRNA expression levels of eight organizations (foregut, midgut, hindgut, liver, spleen, kidney, heart and gill) were quantitatively studied.4. The response of Cyprinus carpio L. PepTl gene relative expression abundance to different concentrations of oligopeptide. In the present study,5%,10%,15%oligopeptide preparation were added respectively in fish feed. The results showed that there was a positive response of the PepT1mRNA expression to the upward of the oligopeptide addition amount. PepTl mRNA expression in midgut was significantly changed with the oligopeptide gradient.