真鲷天然抗性相关巨噬蛋白全长cDNA的克隆与序列分析

Cloning and sequence analysis of Nramp cDNA from Pagrus major

Natural resistance associated macrophage protein (Nramp) is an innate resistance protein to intracellular parasites, which is expressed plentifully in macrophage cells. Nramp has been studied in mouse, human, cattle, rainbow trout and channel catfish. However, little was known about the structure of Pagrus major Nramp. In order to get the complete sequence of Pagrus major Nramp, a pair of primer is designed according to a 200bp known sequence of Pagrus major Nramp cDNA. By the use of SMART RACE, the full Nramp of Pagrus major cDNA about 5 000 bp was obtained, including about 200 bp 5′ terminal region (UTR), complete encoding region and 3′ terminal region. There were 3 ployA signals, which showed many possibilities of cutting at 3′ terminal region. The character of Pagrus major Nramp nucleotide sequence and deduced amino acid sequence are analyzed. 12 putative transmembrane(TM) regions, a consensus transport motif (CTM), a predicted protein kinase C phosphroylation site and three predicted N-link glycosylation sites are indicated in its deduced amino acid sequence. The ’consense transport motif’ CTM is located between TM8 and TM9. Furthermore, a protein kinase C phosphroylation site and three N-link glycosylation sites were predicted. The alignment of amino acid sequences between Pagrus major Nramp cDNA and several animals is analyzed and the deduced amino acid sequence of Pagrus major Nramp had 77.8%, 83.0%, 82.3%, 80.0%, 81.1%, 60.4%, 70.3%, 58.5%, 69.5% identity with rainbow trout α(AAD20721), rainbow trout β (AAD20722), channel catfish(AF400108), fathead minnow (AAF01778), common carp (CAB60196), mouse 1(AAA39838), mouse 2(AAC42051), human 1(D50403), human 2(NP-000608), respectively. The alignment reveals high conservation in TM and CTM regions. Analysis result makes us get familiar with the structure and character of fish Nramp, furthermore, offers some information for the enhancement of immunity of fish and genetic amelioration on fish breeding.