Construction of a cDNA library and cloning of the MHC class I gene in silver carp (Hypophthalmichthys molitrix) infected with bacterial septicemia

Abstract

Silver carp(Hypophthalmichthys molitrix) is one of the most commonly cultured freshwater species in China.However,the development of silver carp aquaculture is threatened by bacterial septicemia.To isolate and study genes relevant to the disease,we constructed a cDNA library from silver carp liver and kidney tissue using a CloneMiner? cDNA kit.The primary cDNA library titer was 1.34×107 cfu/mL yielding 2.68×107 cfu recombi-nants with 97.5% positive clones.The exogenous inserts of the recombinants ranged in size from 0.8 to 3.5 kb.We attempted to sequence 80 positive clones from both terminals to test the completeness of the coding sequence.We successfully sequenced 74 clones,of which 49 contained the complete coding sequence.Among the clones suc-cessfully sequenced,57 sequences were identified to 49 known genes in GenBank.The cDNA library was subse-quently screened by PCR yielding a single clone containing the complete coding sequence for MHC class I.The H.molitrix MHC class I was 1 026 bp long and encoded a 341 amino acid(aa) protein that included a leader peptide,α1,α2,α3,and transmembrane and cytosolic domains of 16,88,90,87,and 60 aa,respectively.In addition,we identified sites that were highly conserved among vertebrate MHC class I.Phylogenetic comparison of the com-plete coding sequences and the α3 domain of H.molitrix MHC class I with other vertebrate species revealed dif-ferent topology,suggesting a different evolutionary history for different domains of MHC class I and the occur-rence of gene recombination among cyprinidae.