Cloning, Expression And Polyclonal Antibody Preparation Of Major Histocompatibility Complex Class Ⅱα,β Genes Of Duck

The major histocompatibility complex(MHC) is an extended cluster of genes with extraordinary polymorphism and compact linkage in the chromosome,it encode immunoglobulinlike receptors and spreading over all vertebrates,which strongly associated with disease resistance and immune response.According to their structure and function,MHC molecules could be divided into classâ… ,â…¡andâ…¢.MHCâ…¡molecules are glycoproteins that are heterodimers consisting ofαandβchains.MHCâ…¡molecules mainly distribute on the membrane of the maturing B cells,presenting cells(macrophage cells and dendritic cells) and activated T cells.These molecules have an effect on presenting antigen and activating T cells by forming classâ…¡molecule-antigen peptide complex, which can stimulate T cells and B cells to participate in immune response.It has an important role in humoral or cell-mediated immune response.Weather the expression or expression level of MHCâ…¡molecules,which determin the strength of immune response.Moreover,the changes of MHCâ…¡molecules expression level which participate in some autoallergic disease,tumor and immune deficiency etc disease's development.So it has an significant in immunology research.In order to research the function and mechanism of class MHCâ…¡of duck in immune response,we cloned, expressed and preparation the polyclonal antibody.Firstly,according to mRNA gene sequence registered in GenBank and multi-cloing restriction enzyme sites of vector,two pairs of specific primers for the genes of chainαandβof classâ…¡of duck were designed and synthesized respectively.Using total RNA from duck spleen,two genes about 633 bp and 798 bp were amplified by RT-PCR respectively.Then cloned into the T cloning vector pMD18-T.After being identified by restriction digestion and DNA sequencing,the inserts were subcloned into the expression vector pET-28a and pGEX-KG respectively.The results showed we cloned the MHCⅡαand MHCⅡβgene,the obtained 633 bp fragment of MHCⅡαgene has 99% identities to the previously identified duck MHCâ…¡alpa at nucleotide level;Meanwhile,the obtained 798 bp of MHCⅡβgene has 92%identities to the previously identified duck MHCâ…¡beat at nucleotide level.Secondly,the constructed recombinant plasmids pET-28a-αand pGEX-KG-βwere transformed into E.coli BL21(DE3) and then induced to express MHCⅡαand MHCⅡβfusion protein by IPTG.After optimizing prokatyotic expression conditions which include induction temperature and time, concentration of inductor,initiatal OD₆₀₀ value,ventilatory capacity,pH of medium and concentration of antibiotic.We definited at 22℃,induce 3 h,0.05 mmol/L 1PTG,initiatal OD₆₀₀ value 0.45, ventilatory capacity 70%,pH 7.2,50μg/mL Kna are the best conditon for expression MHCⅡαfusion protein,and MHCⅡβfusion protein at 37℃,induce 3 h,0.50 mmol/L IPTG,initial OD₆₀₀ value 0.45, ventilatory capacity 70%,pH 5.7,50μg/mL Amp can expressed efficiently.The expressed MHCⅡαand MHCⅡβfusion protein were located in inclusion bodies.The obtained MHCⅡαand MHCⅡβ inclusion body proteins were solubilized by sonication with the adding of the detergent lauroylsarcosine.The solubilized MHCⅡαand MHCⅡβprotein purified by affinity chromatography and the purity reached above 90%.Finally,mice were immunizated with the purified MHCⅡαand MHCⅡβfusion protein and the polyclonal antiserum against chainαandβof duck were collected.A specific reaction appeared between the antibodies and MHCⅡαand MHCⅡβfusion protein in ELISA and Western blot,these results indicated that fusion proteins,MHCⅡαand MHCⅡβfusion protein,had strong antigenity.In conclusion,in this study we successfully cloned the genes of chainαandβof duck MHCâ…¡molecules,constructed the prokaryotic expression vector and the protein expressed efficiently. Prepared high titer and specific antibodies.All these would provide some exprimental materials for the futher studies on the immunity function ofαandβof duck MHCâ…¡molecules.