Cloning And Prokaryotic Expression Of Chicken MHCⅡα/β Chain And Preparation Of Their Polyclonal Antibodies

The major histocompatibility complex (MHC) is an extended cluster of genes with extraordinary polymorphism and compact linkage in the chromosome, spreading over all vertebrates. MHC molecules are also considered as the primary determinants in the transplant rejection. According to their structure and function, MHC molecules could be divided into class Ⅰ, Ⅱ and Ⅲ. Class Ⅱ molecules are glycoproteins that are formed by two noncovalence-associated chains, a (32000~34000) and p (29000~32000), each of which is encoded by different MHC genes. Like class I molecules, class II molecules consist of peptide-binding region, immunoglobulin-like region, transmenbrane region and cytoplasmic region. Class Ⅱ 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 class II molecule-antigen peptide complex, which can stimulate T cells and B cells to participate in immune response. However class Ⅱ molecules mostly present exogenous antigen. In order to research the function and mechanism of class MHC Ⅱ of chicken in immune response, we cloned, expressed and identified the gene of chain α and β of class Ⅱ.Firstly, according to mRNA gene sequence registered in GenBank and multi-cloning restriction enzyme sites of vector pGEX-4T-1, two pairs of specific primers for the genes of chain α and β of class II of chicken were designed and synthesized respectively. Using total RNA from ConA-stimulated chicken spleen lymphocytes, two genes about 771bp and 798bp were amplified by RT-PCR respectively. The results of endonuclease EcoRI digesting RT-PCR product of chain a and endonuclease PstI digesting RT-PCR product of chain β show that two genes have the corresponding restriction enzyme site as the known α and β gene. Then the genes were inserted into vector pGEX-4T-1 respectively, and the recombinant plasmids were transformed into E.coli BL21. The recombinant plasmid pGEX-4T-1/α was identified by BamHI+SalI digestion and the recombinant plasmid pGEX-4T-l/p was identified by EcoRI+SalI digestion. Then, the positive recombinant clone was sequenced and analyzed. The results show that the complete ORF of a gene contains 771 nucleic acids and encodes 256 amino acids; the nucleotide sequence similarity is approximately 99% with the reported a gene. Meanwhile, the complete ORF of β gene contains 798 nucleic acids and encodes 266 amino acids; the nucleotide sequence similarity is approximately 95% with the reported chicken β gene. These suggest that the aand β genes of chicken MHC class II molecules have been successfully cloned from chicken spleen lymphocytes.Secondly, the constructed recombinant plasmids pGEX-4T-1/α and pGEX-4T-l/p were transformed into E.coli BL21 and then induced to express GST-a and GST-P fusion protein by IPTG at different concentration and at different times. After optimizing prokaryotic expression conditions, 4 h was determined as the optimum induction time and 1 mmol/L was determined as the concentration of IPTG. GST-a and GST-P fusion protein solubilities were identificated and the result indicated that expressed GST-a and GST-P protein were located in inclusion bodies. Under the optimal condition, the recombinant plasmids were induced to express large-scale GST-a and GST-P fusion proteins and the induced recombinant bacteria were lysed by freeze-thaw and sonication. The obtained GST-a and GST-P inclusion body proteins were solubilized by sonication with the adding of the detergent lauroylsarcosine. The solubilized GST-a and GST-p protein were purified by affinity chromatography with glutathione agarose.Finally, mice were immunizated with the purified GST-a and GST-P fusion proteins and the polyclonal antiserum against chain a and p of chicken were collected. A specific reaction appeared between the antibodies and GST-a and GST-P fusion protein in agar diffusion assay and in ELISA, these results indicated that fusion proteins, GST-a...