Cloning And Function Of The Major Histocompatibility Complex Class Ⅱ Genes’ CDNA Of Chinese Sturgeon (Acipenser Sinensis)

Chinese Sturgeon（Acipenser sinensis）is an ancient and precious fish of cartilage and ganoin.It is called"living fossil in water",as it is of great significance for research on the biological evolution.At present,the research on Chinese sturgeon is mainly focused on artificial breeding,but only few studies on the immunology of Chinese sturgeon were carried out.The major histocompatibility complex Ⅱ（MHC Ⅱ）molecule plays an important role in adaptive immune responses by presenting antigenic polypeptides to CD4+T lymphocytes.MHC Ⅱ is a heterodimer composed of oneαsubunit and oneβsubunit.It’s correct and efficient transported need the help of the Ii chain（MHC Ⅱ-association invariant chain,also known as MHC Ⅱγ）.Here,research on the molecular cloning of Chinese Sturgeon MHC Ⅱ（CsMHC Ⅱ）cDNA and it’s immunological function was performed as follows:1.Total RNA was extracted from the spleen tissue of Chinese sturgeon and a cDNA library was successfully constructed by RT-PCR.The cDNAs of CsMHC Ⅱα,βandγchains were identified by randomly sequencing the cDNA library.Results showed that the cDNA sequences of CsMHC Ⅱα,CsMHC Ⅱβand CsMHC Ⅱγcontained open reading frames of 726 bp,798 bp and 879 bp,respectively.The amino acid sequence comparison results show that CsMHC Ⅱα/βcontains almost all the characteristic features of MHC Ⅱα/βsequences known to other species:a leader peptide,α1/α2 orβ1/β2 domains,CP/TM/CYT region and conservative Cystine residues.Similar to other fish MHC Ⅱαchains,CsMHC Ⅱαalso contains one potential N-glycosylation site(³⁶NGT³⁸)located inα1 domain of CsMHC Ⅱα,while it is usually present inα2domain of other fish MHC Ⅱαchains.There were usually two potential N-like glycosylation sites located inα1andα2 domains in mammalian and amphibian MHC Ⅱα,but only one in fish CsMHC Ⅱα.CsMHC Ⅱγcontains an Ig-MHC like region,class Ⅱ-associated invariant chain Ii peptide（CLIP）,thyroglobulin type-1 repeat region and three conserved disulfide bonds,along with two potential N-glycosylation sites(¹⁵²NET¹⁵⁴and ¹⁶⁷NSS¹⁶⁹).2.Expression of MHC Ⅱα,βandγin Chinese sturgeon tissues was detected by qRT-PCR.Results showed that the highest expression was found in spleen for MHC Ⅱαandβchains,while in head kidney for MHC Ⅱγchain.Stimulation to Chinese sturgeon with inactivated trivalent bacterial vaccine or polyinosinic polycytidylic acid（poly（I:C））up-regulated the expressions of MHC Ⅱα,andβmRNAs,although their transcripts were overall more quickly up-regulated by poly（I:C）than by bacterial vaccine.However,poly（I:C）induced higher MHC Ⅱγexpression than bacterial vaccine in intestine and spleen,while lower than bacterial vaccine in head kidney and liver.3.We constructed the pCMV-His-CsMHC Ⅱα,pCMV-HA-CsMHC Ⅱβ,pCMV-Flag-CsMHC Ⅱγand pCMV-Flag-mutCsMHC Ⅱγ(Ser²⁹/Ser³⁴→Ala²⁹/Ala³⁴)expression plasmid.When co-expressed in mouse dendritic cells,the CsMHC Ⅱγchain bound to both the MHC Ⅱαandβchains.Furthermore,the over-expressed CsMHC Ⅱγchain,not CsMHC Ⅱαor CsMHC Ⅱβchain significantly,activated NF-κB and STAT3 in mouse dendritic cells,and induced downstream gene TNF-αand IL-6expressions as well.This activity was nearly abolished by mutation of the Ser²⁹/Ser³⁴ to Ala²⁹/Ala³⁴ in CsMHC Ⅱγ.This research showed that the MHC Ⅱα,β,γchain of Chinese sturgeon played a important role in the immune response to the pathogenic microorganisms infection of Chinese sturgeon,possibly through the conservative functional mechanisms during process of vertebrates evolution.This will contribute to understanding the immunobiological characteristics of Chinese sturgeon,and provide theoretical and experimental guidance the prevention and control of the disease during the breeding and protection of Chinese sturgeon.