Cloning, characterization and expression analysis of SIMP (source of immunodominant MHC-associated peptides) in grass carp Ctenopharyngodon idella.
SIMP (source of immunodominant MHC-associated peptides) plays a key role in N-linked glycosylation with the active site of oligosaccharyltransferase, being the source of MHC-peptides in the MHC I presentation pathway. In the present study, the SIMP gene has been cloned from grass carp Ctenopharyngodon idella by rapid amplification of cDNA ends (RACE). The full length of the cDNA sequence is 4384bp, including a 1117bp 5' UTR (untranslated region), a 2418bp open reading frame, and a 849bp 3' UTR. The deduced amino acids of the grass carp SIMP (gcSIMP) are a highly conserved protein with a STT3 domain and 11 transmembrane regions. The gcSIMP spans over more than 24,212bp in length, containing 16 exons and 15 introns. Most encoding exons, except the first and the 15th, have the same length as those in human and mouse. The gcSIMP promoter contains many putative transcription factor binding sites, such as Oct-1, GCN4, YY1, Sp1, Palpha, TBP, GATA-1, C/EBP beta, and five C/EBP alpha binding sites. The mRNA expression of gcSIMP in different organs was examined by real-time PCR. The gcSIMP was distributed in all the organs examined, with the highest level in brain, followed by the level in the heart, liver, gill, trunk kidney, muscle, head kidney, thymus, and the lowest level in spleen. Furthermore, the recombinant gcSIMP has been constructed successfully and expressed in Escherichia coli by using pQE-40 vector, and the polyclonal antibody for rabbit has been successfully obtained, which was verified to be specific. Identification of gcSIMP will help to explore the function in fish innate immunity.
Xu
Z Y
ZY
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, People's Republic of China.
Nie
P
P
Chang
M X
MX
Sun
B J
BJ
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D002347:Carps / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D016678:Genome; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment
D011994:Recombinant Proteins; D016695:Glycosyltransferases