Cloning, Expression, And Polymorphic Analysis Of Mhc II B And Cathepsin D Genes Of Grass Carp

Grass carp (Ctenopharyngodon idellus) the third biggest contributor to theworld’s aquaculture production, used in this experiment. Major histocompatibilitycomplex (MHC) class II molecules play a key role in the immune system ofvertebrates. cathepsin D is a lysosomal aspartic proteinase which participates invarious degradation functions within the cell. The research studied the cloning,polymorphism, and the expression of MHC II B and cathepsin D by RACE-PCR,cloning and Real Time PCR from grass carp. These results are shown as follows:We present full-length MHC II B cDNA and genomic sequences of grass carp,The full-length cDNA (950bp) contained750bp open reading frame (ORF), whichencoded a predicted protein with250amino acid residues. The genomic sequence wasfurther identified to be2207bp in length, which contained six exons and five introns.Interestingly, the133bp exon4in grass carp MHC II B is longer than that (114bp) ofother fish species, which makes the grass carp MHC II B stop codon locate in exon4,not in exon5. The deduced amino acid sequence had a22.8-56.3%identity with thoseof other species. Four domains of grass carp MHC II B protein were revealed: a signalpeptide from1to25, a beta domain from31to106, and immunoglobulin c-type1(IGc1) from131to202, and a transmembrane domain from221to243.In this current study, we cloned and characterized the complete cDNA of grasscarp cathepsin D through5′-and3′-RACE. The cathepsin D contained a56bp5′terminal untranslated region (5′-UTR), a1197bp open reading frame encoding398amino acids, and a394bp3′-UTR. Unlike many other organisms, the grass carpcathepsin D contains only one N-glycosylation site closest to the N-terminal. According to the ProtParam program predication, the grass carp pre-pro-cathepsin Dmass is43105.50Da and theoretical isoelectric point is5.67.The molecular weight ofthe mature peptide is36411.62Da and the pI is5.06. Grass carp mature peptide wasclassified as stable protein for its instability index (II) is35.24. The total number ofnegatively (Asp+Glu) and positively charged (Arg+Lys) residues was32and24,and the grand average of hydropathicity (GRAVY) of the mature peptide is-0.02, so itwas classified as hydrophilic protein.One N-linked glycosylation site (N-X-S/T) was respectively identified in thebeta-1and beta-2domain, which is different from that of other mammals and teleost(only one N-linked glycosylation site locating in the beta-1domain). Six differentalleles were identified from seven individuals. There was evidence of strong positiveselection at beta-1PBR positions (dN/dS=2.3, P <0.05) between grass carpdifferent sequences, and a slight but non-significant indication of selection atnon-PBR beta-1region, beta-2region.The MHC II B expression showed an downward at the begging of the embryonicdevelopment and then upward trend at the late of embryonic development. Real-timequantitative revealed the high expression of MHC II B in head-kidney, spleen, gill,intestine, and brain, and lower expression in live, heart, fin, and other tissues. Thepresent findings will provide valuable information for understanding structure,function, and molecular polymorphism of MHC II B in grass carp. Real-timequantitative RT-PCR demonstrated that cathepsin D expressed in all twelve tissues(bladder, brain, liver, heart, gill, muscle, fin, eye, intestines, spleen, gonad and headkidney). The relative expression levels of cathepsin D in gonad and liver were26.58and24.95times as much as those in fin, respectively. The expression level ofcathepsin D in muscle approximately16-fold higher, in intestines and spleen were12-fold higher. The cathepsin D expression showed an upward trend duringembryonic development.After challenged with Aeromonas hydrophil, the expression of grass carp MHCII B gene showed significant changes in liver, head kidney, spleen and intestines Thefact that the bacterial infection can improve the MHC II B andcathepsin D gene expression in immune-related organs, may suggest that MHC II B and cathepsin Dplay an important role in the innate immune response of grass carp.