β₂m CDNA Cloning, MHC Class Ⅰ Diversity And Its Recombinant Protein Identification Of The Grass Carp

In order to clarify molecular characteristic of grass carp MHC I (Ctid-MHC I ) and β₂m (Ctid-β₂m), and to promote the research of MHC binding with antigen peptide in low order vertebrates, the Ctid-β₂m gene was cloned and the diversity of Ctid-MHC I was analyzed. At the same time, Ctid-MHC I expression amount from different tissues was determined. Finally, recombinant protein of Ctid-MHC I was expressed and identified.Firstly, a grass carp kidney cDNA PCR Library was constructed. Based on the sequences alignment of other fishes, degenerate primers of grass carp MHC I light chain were designated to amplify Ctid-β₂m. 5' from the library, and then Ctid-β₂m full cDNA was cloned by anchored-PCR. Secondly, Ctid-MHC I genes from 15 individuals were isolated by RT-PCR. At the same time, a prokaryotic expression system of pMAL^TM-p2X was used to express Ctid-MHC I . Finally, based on the diversity investigation of the Ctid-MHC I , a quantitative competitive reverse transcription PCR (QC-RT-PCR) was designed to analyze the Ctid-MHC I expression in different tissues from the same individual.Results show that the cloned Ctid-β₂m codes for a 116 amino acids peptide, including 19 amino acids of signal peptide and 97 amino acids of mature protein. It shares a 58.6%-75.0% aa homology with that of other bony fishes. According to the sequences analysis, Ctid-β₂m can be grouped into two lineages (β₂m-NA, β₂m-NB). The two lineages share an amino acids homology of 94.8%, and one of them has a potential N-glycosylation site at position 107-109. Fifteen different Ctid-MHC I genes were screened out by RT-PCR, and 13 of them shared the same 8 critical amino acids binding with antigen peptide in other fish species (YYRTKWYY). Alignment of all the known Ctid-MHC I can be grouped into eight lineages (Ctid-NA-Ctid-NH). Domains homology BLAST and phylogenetic tree constructed with other teleosts show that Ctid-MHC I α1, α2 and α3 can be categorized into two (V and IX), five (II, IV-VII) and four (IV-VII) domain lineages, respectively. Among them, one novel al(IX), four new α2 (IV, V, VI, VII), and three unique α3 (V, VI, VII) domain lineages are only observed in grass carp. QC-RT-PCR resulted in a different expression amount of Ctid-MHC I among kidney, gill, intestine, heart, spleen, liver and brain. SDS-PAGE and Western blot show that the recombinant protein of Ctid-MHC I has a molecular weight of about 78.68kD.The research shows the clear characters of Ctid-MHC I and Ctid-β₂m, and pushes an advance on the research of fish MHC. The data also provide information for further studies on anti-disease breeding and epitope vaccines.