The Role Of Major Histocompatibility Complex Class Ii Molecule In Mucosal Immunity In Common Carp (cyprinus Carpio L.)

The major histocompatibility complex (MHC) is a genomic region that encodes cellular proteins that are involved in the immune response in vertebrates. The MHC class II molecule, consisting of one alpha chain and one beta chain, present foreign peptides derived from extracellular pathogens and present them to CD4+T cells, and triggering a specific immune response towards the pathogen from which the peptides are derived in the adaptive immune system.Partial cDNA of carp MHC class IIβwas amplified from the spleen of carp (Cyprinus carpio) using RT-PCR. The cDNA is 610 bases and encodes 202 amino acids, which contain partial leader peptide,β1 domain,β2 domain and partial connecting peptide.Amino acids sequence of carp MHC class IIβis highly similar to other teleost fishes but less similar to mammalian and shark. In teleost, the similarity of theβ2 domain is higher thanβ1 domain. A phylogentic tree was constructed with the amino acid sequence of these MHC class IIβ, indicating that two clusters were present: one is mammalian and amphibian MHC class IIβ, the other is fish MHC class IIβ. It is obvious that the carp MHC class IIβis more similar to that of Danio rerio.RT-PCR demonstrated that carp MHC IIβtranscripts were highly abundant in head kidney, spleen, skin, foregut, hindgut, oral epithelial, gill, gonad and whole blood, less abundant in liver and muscle.The kinetics of MHC class IIβexpression after stimulation was similar in spleen, head kidney, foregut and hindgut although the fold induction was different in these tissues. The highest induction level in spleen and foregut was apparently at day 3 and was followed by a decline until day 7 when the MHC class IIβexpression in In-L.ang-injected fish was comparable to the control fish. In head kidney and hindgut, the highest induction level was at hour 6.The extracellular part of carp MHC class IIβwas cloned by using the methods of molecular cloning and recombinant technics, and connected to pET-28a(+) vector. E.coli BL21 (DE3) was transformed with the recombinant expression vector pET-28a(+)/MHC II B and expressed the recombinant protein in adding of 1mM IPTG at 37℃. SDS-PAGE results showed that a protein with a molecular weight of 28.9 kD was expressed within the inclusion bodies. The protein was used as antigen to immune Zealand male rabbits for l month to gain the rabbit-anti-carp MHC class IIβchain antiserum. In order to get the mouse antisera against recombinant carp MHC class IIβchain, we designed the DNA immunity experiment. Firstly, we designed the MHC class IIβsegment with Kozak and ATG sequences and constructed the pcDNA3.1(+)/MHC II B vector. Then the highly purity recombinant vector was extracted and injected to mice to get the mice-anti-carp MHC class IIβchain antiserum.The recombinant protein expressed in E.coli BL21 (DE3) was used to test the identity of rabbit-anti-carp and mice-anti-carp MHC class IIβchain antisera by western-blot experiment. The titers of these antisera were 1:32 and 1:16 according to the double agar diffusion experiments.These antisera were obtained and used for the immunohistochemistry of MHC class IIβchain. The results showed that the MHC class IIβchain of carp was located in intestinal epithelial cells and lymphoid cells.