藏猪兴奋性氨基酸转运载体EAA C1基因的克隆与原核表达

摘要

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 EAA C1 in pigs.%兴奋性氨基酸转运载体EAAC1是肠道内谷氨酸的主要载体.本研究根据人的基因编码区设计一对特异性引物,以藏猪空肠总RNA为模板,通过RT-PCR获得一长约1600 bp的cDNA片段,T/A克隆后测序,并进行序列分析；将该基因片段连接到原核表达载体pET-32a(+)中构建融合表达质粒,转化到E.coli BL21(DE3)中进行表达.测序结果显示,获得的cDNA全长为1575 bp,编码524个氨基酸；EAAC1分子量为57 kD,等电点为5.34,GenBank登录号为GQ375513;该蛋白质具有3个N-糖基化位点、8个蛋白激酶C磷酸化位点和1个cAMP/cGMP依赖性蛋白激酶磷酸化位点,含有7个跨膜结构域和一个信号肽序列；SDS-PAGE电泳检测结果表明,表达蛋白质与预期蛋白质大小一致.上述结果为进一步研究EAAC1的功能及其调控奠定了基础.