Cloning, Expression And Antibody Preparetation Of Porcine CD8a Genes And Cloning And Identification Of Pigeon Ii Gene

CD8 was typeâ… integral membrane proteins mainly expressed at the surface of T helper lymphocytes(Th)and cytotoxic/suppressor T lymphocytes(CTL/Ts).CD8 has been shown to function in mediating signal transduction and adhesion on a subset of cells within the T cell compartment and is critically involved in the development of T cells expressing MHC classâ… -restricted T cell receptor(TCR).CD8 genes are selectively expressed on the surface of TCRaβT cells,thymocytes,intestinal intraepithelial lymphocytes and natural killer cells.CD8a is a transmembrane glycoprotein with an N-terminal Ig-like domain,a hinge region,and a cytoplasmic domain responsible for interaction with p56^lck,the lymphocyte specific kinase.Given the importance of knowing the molecular interactions between the CD8 and MHC molecules,we expressed the largest ORF of porcine CD8a.Firstly,specific primers for porcine CD8a were designed from the putative coding regions of the porcine CD8a registered in GenBank,total RNA was prepared from Concanavalin A-stimulated porcine spleen according to the instructions of the TRIzol reagent,one gene fragment was amplified by reverse transcription-polymerase chain reaction,the results of endonuclease digesting RT-PCR products showed that the gene fragment have the same restriction enzyme sites as known pCD8a.Subsequently,gene fragment was ligated to pMD18-T vector after purified,the recombinant plasmid pMD18-T-CD8a was transformed into E.coli BL21 by standard methods,the recombinant plasmid was sequenced and analysed after they were identified by PCR.The results suggest that the obtained gene fragments contain 711 bp.The results indicated that the pCD8a have been successfully cloned from porcine spleen.After sequencing,the CD8a cDNA sequences were aligned using the JELLYFISH program.The cloned porcine CD8a shared 99.2%identity with the previously identified pCD8a at nucleotide level.Secondly,gene-specific primers were designed based on the cloned CD8a nucleotide sequences and multi-clone restriction enzyme sites of prokaryotic expressive vector pGEX-4T-1,the pCD8a was amplified from recombinant plasmid pMD18-T-CD8a by PCR,and it was 720bp in length.Then the cDNA fragments were cloned into pGEX-4T-1, the recombinant plasmid were identified by PCR and endonucleases EcoRâ… & Salâ… digestion.The recombinants,namely pGEX-4T-1-CD8a,were transformed into E.coli strain BL21 and GST-CD8a fusion proteins were induced to express.The MW of the fusion proteins was about 52 KD as analyzed by SDS-PAGE.And the solubility of fusion proteins was identified,the expressed fusion protein GST-CD8a located in inclusion bodies.Finally,after optimizing prokaryotic expression conditions,we determined the optimum inducement time and concentration of IPTG.The recombinant plasmids pGEX-4T-1-CD8a was induced to express large-scale fusion protein GST-CD8a,the induced recombinant bacteria was lysed by freeze-thaw and sonication.At last,we obtained the GST-CD8a inclusion body protein,which could be solubilized by sonication after the detergent lauroylsarcosine was added.The reconstructed porcine CD8a prokaryotic expression plasmid was applied to express successfully in E.coli.And the anti-CD8a multi-antibody that we prepared by the fusion protein can be applied for further research.In order to examine the expression of the porcine CD8a protein,we developed a multi-antibody to porcine CD8a fusion protein.This fusion protein was injected into mice and anti-porcine CD8a sera were obtained.Western blot analyses shows that one hybridization band of about 52kD was found from the recombinant protein.it suggested that the polyclonal antibody was specific for CD8a.In conclusion,we cloned and expressed CD8a gene in prokaryocyte and generated mice anti-porcine CD8a multi-antibody successfully.All these provide some experimental materials for future studies on the immunity function of porcine CD8a. Invariant chain was a nonpolymorphic typeâ…¡transmembrane glycoprotein,which mainly expresses on the surface of B cells,macrophage cells,dendritic cells and thymic epithelial cells etc.As a chaperone of MHCâ…¡molecule,Ii mainly has the following functions:(1)It can facilitate the proper fold of the MHCâ…¡heterodimeric and egress from the endoplasmic reticulum(ER);(2)It can provide a targeting signal for endosomal/ lysosomal compartments;(3)It prevents peptide from associating prematurely with MHCâ…¡molecule.So Ii plays a key role in MHCâ…¡molecular presenting exogenous antigen to CD4⁺ T lymphocytes and has an important regulated significance on immune reaction.According to homology sequence from chicken,duck and so on,by means of designing degenerate primer,we gained the high conserved domain of pIi gene cDNA from pigeon spleen firstly.The 3' terminal of pigeon Ii(pIi)gene cDNA was cloned by 3'-RACE technique based on the sequencing rusults of first step.Then the 5'terminal of pIi gene cDNA was cloned by 5'-RACE technique in the same way.According to the results of 3'-RACE and 5'-RACE we design a pair of primers for the item to clone the full length of pIi gene.Finally,the pIi gene cDNA is 1050bp long with an open reading frame of 633bp encoding for a protein of 211 amino acids was cloned successfully.The access number in Genbank is AY904337.The comparison of the deduced amino acids testified that pIi gene contain a 26 amino acid variant CLIP(classâ…¡-associated invariant chain peptides)domain which combining with MHCâ…¡protein,a TRIM domain which helps Ii synthesis into trimerizes,and most key residues proposed important for the biological function were conserved with other species.We have successfully cloned the pIi gene and analysied the deduced amino acids sequence and function domain of pIi.All these provide some experimental materials for the future studies on the immunity function of pIi.