Cloning and expression analysis of histocompatibility complexⅠα antigen (MHCⅠα)from humphead snapper Lutjanus sanguineus

In the present study,full-length cDNA sequences of histocompatibility complexⅠα antigen(MHCⅠα)was cloned by rapid amplification of cDNA ends technique (RACE)from humphead snapper(Lutjanus sanguineus).Full-length cDNA sequence of MHCⅠα is 1 369 bp,encoding 354 amino acids.BLAST analysis revealed that the amino ids sequence of MHCⅠα shared high identity(84%)with other MHCⅠ.Phylogenetic tree was constructed by the Neighbor-Joining method,and the results suggested MHCⅠα of humphead snapper shared the closest genetic relationship with the MHCⅠof Epinephelus coioides.The results of fluorescent real -time quantitative RT-PCR showed that the expression of MHCⅠα mRNA could be detected in head kidney,and the maximum expression appeared in 6-15 h post infection.MHCⅠα was subcloned into pET32a(+)to construct expression plasmids pET32-MHCⅠα.SDS-PAGE and Western blot analysis indicated that the recombinant proteins were successfully expressed in Escherichia coli BL21(DE3).Then the recombinant proteins were purified and the antiserum was obtained by immunizing rabbits with the purified recombinant proteins emulsified with adjuvant.ELISA analysis showed that the titer of the antiserum prepared in this study was 1∶40 000.The results of the Western blot revealed that specific antigen-antibody reaction occurred between the antiserum and the recombinant proteins.