Characters Of Binding Activity Of Chicken B-L And Ii Gene Expressed Products

Major histocompatibility complex(MHC)is some protein molecules encoded by a closely linked gene group located in vertebrate genomes,which appear high degree of polymorphism.MHC molecules distribute on various cell surface,present antigen peptides and regulate immune response.In the different animals it and its encoding products are given special names.In MHC molecular family class Ⅱ is consisted of two chains,α and β,as a heterodimers.In endoplasmic reticulum it binds invariant chain(Ii)to form nonamer.Ii is a type Ⅱ transmembrane glycoprotein,its leukocyte differentiation antigen is CD74.Ii assists MHC Ⅱ molecules to correct folding,combination,structure maintains,present exogenous antigen peptide.The CLIP region(the class-Ⅱ associated Ii chain peptide)in Ii occupies the antigen binding groove of MHC Ⅱ molecules during aggregation α,β chains to form polymer,which prevents endogenous peptide from associating with the MHC Ⅱ class of molecules.The research of an interaction between Ii with MHC molecules is via in eukaryotic and prokaryotic expression system.In the study of their interaction there is an analysis of the affinity between both molecules,but the eukaryotic expression system can not need to a large number of expressed products.Therefor in this study the prokaryotic expression system was used to detect the interaction between the both molecules.First,chicken B-LB(login ID: S66480)gene sequences in Gen Bank was achieved.With a series of self-designed primers,genes of B-LB β1β2 coding region were cloned from the cDNAs saved in our laboratory respectively.These genes were further inserted into prokaryotic expression carrier p ET-28 a,respectively,which were used to transfect E.coli,Rosetta(DE3).The result of identification by PCR,ouble-enzyme cleavage and sequencing showed that three recombinant plasmids were successfully formed.Secondly,prokaryotic expression products were repeatedly frozen and thawed,and splitted with ultrasonic treatment respectively.Then the samples were dialysis after refolded and purified with Ni Sepharose TM 6 Fast Flow(His/B-LB-26-209、218、223).The results of SDS-PAGE showed that molecular weights of purified proteins were26.4kD(His/B-LB-26-209),27.4 kD(His/B-LB-26-218),28.0 kD(His/B-LB-26-223),respectively,which was consistent with the expected value.These purified prokaryotic expression proteins were then detect in a western blot.The results indicated that the prokaryotic expression proteins had antigenicity.Finally,the dissociation constants(Kd),reaction enthalpy and entropy changes(△H,△S)of B-L with Ii were detected by the ITC.The results showed that B-L with Ii are mainly controlled by the electrostatic attraction and hydrogen bonding.All of these indicated that the prokaryotic expressed products of chicken B-L genes could retain their antigenicity,and in Isothermal Titration Calorimetry method the prokaryotic expressed chicken B-Land Ii molecules could bind to form complex and show the affinity after their renaturation.The results in this work provide a useful method to study the relation between Ii and MHC molecules.