Gene Cloning and Prokaryotic Expressing of Excitatory Amino Acid Carrier 1 in Tibetan Pig

Excitatory amino acid carrier 1(EAAC1) is the major transporter of glutamate in the intestine. A cDNA fragment about 1600 bp was amplified from the total RNA of jejunum in Tibetan piglets by RT-PCR with a pair of specific primers based on the sequences of human. The recombinant vector was constructed by inserting the cDNA fragment into the prokaryotic expression vector pET-32a（＋） and then transformed into E. coli BL21(DE3). Sequence analysis showed that the ORF of EAAC1 cDNA in Tibetan piglets was 1575 bp in length encoding 524 amino acid residues with a molecular mass of 57 kD and isoionic point of 5.34. Its GenBank accession number is GQ375513. Three putative extracellular N-glycosylation sites, eight potential protein kinase C phosphorylation sites and one deduced cAMP/cGMP-dependent protein kinase phosphorylation site were identified. Seven proposed transmembrane domains and a N-terminus signal peptide were presented in EAAC1 sequence. The SDS-PAGE displayed that the expressed protein was consistent with the size of expected protein. These findings maybe provide some scientific basis for the study on function and regulation of EAAC1 in pigs.