Cloning, Expression And Refolding Of Extracellular Region Of Bovine IgG2 Fc Receptor And Crystal Growth

ABSTRACT Immunoglobulin(Ig) Fc receptors(FcRs) expressed on phagocytic cells provide a cruciaLl link between the humoral and cellular branches of the immune system.They have multiple roles in effector responses, antoimmunity to infection, clearance, inflammation and regulation of antibody synthesis.Among the receptors, IgG Fc receptor is the most important kind. Recent studies show that bovine IgG2 Fc receptor (boFc γ 2R) is a new type of mammalian Fc recptor and ruminant Fc recptors play an important super-immune role. So, making out three-dimensional structure of the EC region of bovine IgG2 Fc receptor can demonstrate the structural and functional relation and would be helpful for us to further understand the mechanism that they work in vivo.The intentions of this paper are to clone and express the extracellular region of bovine IgG2 Fc receptor using RT-PCR, and to screen out the situation of crystal growth, so that we can map out the crystal three-dimensional structure of the EC region of bovine IgG2 Fc receptor using X-ray method and demonstrate the mechanism that they work in vivo ultimately.One pair of oligonucleotic primers was designed according to the published sequences of bovine IgG2 Fc receptor gene, which has been published in Genbank with number of Z37506.The extracellular region of 682bp was amplified by RT-PCR from RNA which was extracted from bovine leucocyte.The 682bp fragment was cloned into pGEM-T Easy Vector System I and sequenced. It was found that the fragment has 100% homology with Z37506 in nucleotide and amine acid level, respectively.The extracellular region of bovine IgG2 Fc receptor gene was digested with EcoR I and Not I ,and cloned into pET-28a digested with EcoR I and Not I .The right recombinant plasmid was screened out and identified by enzyme digestion electrophoresis.The special protein was induced by IPTG and expressed ,which is about 30kDa.The denaturalized inclusionbody was refolding and purified, and the...