Gene Cloning, Expression And Bioinformatic Analysis Of Major Histocompatibility Complex Class Ⅱ B From Lampetra Japonica

Lamprey as the most direct ancestor of vertebrates, in order to explore the evolution of vertebrate adaptive immunity provides a wealth of genetic information.The studies of lampre ys immune genes provide the possibility that we understand the origins and evolution of the adaptive immune system.The major histocompatibility complex(MHC) is found in vertebrates encoding immunoglobulin-like receptors of the important non-specific immunity gene. MHC molecules can be divided into MHCⅠ,MHCⅡand MHCⅢthree categories.MHCⅡmolecule s are surface glycoproteins that are heterodimers consisting ofαchains andβchains.There are two kinds of subtypes A and B, MHCⅡB gene are encoding 2 and 3 extrons in the beta 1 and beta 2, even two explicit introns, Their high polymorphism plays an important role in immune response and the research to have the important meaning in the regulation of disease. At present research mainly focuses on MHC in the bony fish and mammals, whether the existence of MHC in Lamprey is unclear. This paper, we the first obtained MHCⅡB gene for its fragments from leucocyte in lamprey by molecular biology methods and bioinformatics techniques.Gene cloning ,expression and bioinformatics analysis,can reveal MHCⅡB genes in lamprey between invertebrates and vertebrates on adaptive immune evolutionary relation ship, as well as further provides theoretical basis, the origins of MHC in the vertebrate and the evolution of classⅡfamily .This research , the first successful clones Lampetra japonicanon-specific immune factors IIB gene cDNA sequence by RACE,semi-quantitativeRT-PCR and bioinformatics technology. The grass for cloning comparison member,MHCstretches for 1209bp,contain 3'UTR, promot er, polypeptide combining area (beta1), IGC area (beta2), transmembrane area, cytoplasmic area and 5'UTR area, coding areas, 834bp size encoding for 277 amino acids ,respectively. Amino acid sequence analysis finds that MHC moleculesⅡB protein in lampetra japonica molecules with classic MHC spatial structure ,the alpha+beta type protein,contains glycosyl site(NST) in beta1 area; the Casein kinaseⅡphosphorylation sites; The beta1, beta2 and transmembrane area contain nutmeg acylation site.In addition, this article compares with 24 different species of MHCⅡB amino acid sequence,and constructs the vertebrates system evol utionary tree.The results show that MHCⅡB sequence of amino acids in lampetra japonica have homocysteine sites for the formation of classic MHCⅡB structure,and the amino acid sequence of beta 2 can be used as the good relationship evolution markup of between vertebrate species.To detemine the MHCⅡB gene expression features in lamprey,we perform the semi-quantitative RT-PCR,the results show the high expression in the gills,heart,blood,with a small amount expression of kidney, but in skin,muscle, liver, intestines, gonad not be expressed.Conclusion:The discovery of MHCⅡB gene in jawless vertebrates (lampetra japonica), for we better understand how the origins and evolution of adaptive immune system provides clues.At the same time to explor the evolution mechanism of MHCⅡB genes laid a foundation.