Cloning, Expression Of Chicken CD4 And CD8α Genes

CD4 and CD8 are type ? integral membrane proteins mainly expressed at the surface of T helper lymphocytes (Th) and cytotoxic/suppressor T lymphocytes(CTL/Ts), respectively. They play a crucial role during antigenic stimulation by major histocompatibility complex (MHC)-bearing cells, CD4 and CD8 have a dual function in this process. First, they act as"assistant"or co-receptor in the function of T cells where specific immunity is mediated by interaction of specific T cell receptor and its ligand peptide MHC molecules, if CD4 and CD8 bind to MHC independently of the TCR, they are defined as adhesion molecules. At present, the co-receptor function theory is dominated in the field. Second, CD4 and CD8 act as signal transduction receptors by triggering the activation of the CD4 and CD8-associated tyrosine kinase p56lck, which modulate, in turn, signaling through the TCR. Whereas the physiological role of CD4 and CD8 remain mostly unknown in p56lck-negative cells, the pathological role for CD4 and CD8αare well documented in all CD4 and CD8-positive cells. Recent studies have also shown that CD4 acts as part of the receptor complex used by HIV to infect its target cell, murine CD8ααbinds to a MHC ?-like thymus leukemia antigen molecule (TL) independently of the TCR. In order to research mechanism of CD4 and CD8 in avian immune defense, in present study chicken CD4 and CD8 were cloned, identified and expressed.Firstly, specific primers for chicken CD4 and CD8αwere designed from the putative coding regions of the chicken CD4 and CD8αregistered in GenBank, total RNA was prepared from Concanavalin A-stimulated thymocytes of a 5-week-old chicken according to the instructions of the TRIzol reagent, two gene fragments were amplified by reverse transcription-polymerase chain reaction (RT-PCR), the results of endonuclease EcoRV and HindⅢdigesting RT-PCR products showed that the two gene fragments have the same restriction enzyme sites as known chicken CD4 and CD8α. Subsequently, gene fragments were ligated to pMD18-T vector after purified, the recombinant plasmids pMD18-T-CD4 and pMD18-T-CD8αwere transformed into Escherichia coli DH5αby standard methods, the recombinants were sequenced and analysed after they were identified by PCR. The results suggest that the obtained gene fragments contain 1380 and 651 base pairs and translate into proteins containing 460 and 217 amino acids, respectively. The cloned chicken CD4 and CD8αshare 99% and 98% identity with the previously identified chicken CD4 and CD8αat nucleotide level, respectively. The results indicated that the chicken CD4 and CD8αhave been successfully cloned from chicken thymus.Secondly, gene-specific primers were designed based on the cloned CD4 and CD8αnucleotide sequences and multi-clone restriction enzyme sites of prokaryotic expressive vector pGEX-4T-1, two gene fragments which encode extracellular domain of CD4 (IgSF D1 and D2) and CD8α(IgSF V) were amplified from recombinant plasmids pMD18-T-CD4 and pMD18-T-CD8αby PCR, they were 579bp and 366bp in length, respectively. Then the cDNA fragments which encode the extracellular domain of CD4 and CD8αwere cloned into pGEX-4T-1, the recombinant plasmids were identified by PCR and endonuclease EcoRⅠ+ SalⅠdigestion. The recombinants, namely pGEX-4T-1-CD4 and pGEX-4T-1-CD8α, were transformed into E.coli strain BL21 and GST-CD4 and GST-CD8αfusion proteins were induced to express. The MW of the fusion proteins were about 47 000 and 39 000 as analyzed by SDS-PAGE, respectively. And the fusion proteins'solubility were identified, the expressed fusion protein GST-CD4 and GST-CD8αlocated in inclusion bodies.Finally, after optimizing prokaryotic expression conditions, we determined the optimum inducement time and concentration of isopropylthio-β-D-galactoside(IPTG). The recombinant plasmids pGEX-4T-1-CD8αwere induced to express large-scale fusion protein GST-CD8α, the induced recombinant bacteria were lysed by freeze-thaw and sonication. At last, we obtained the GST-CD8αinclusion body protein, which could be solubilized by sonication after the detergent lauroylsarcosine was added.In conclusion, we cloned and expressed CD4 and CD8αgenes in prokaryocyte successfully. All these provide some experimental materials for future studies on the immunity function of chicken CD4 and CD8α.