Cloning And Expression Interleukin-2 Receptor Alpha Gene Of Rongchang Porcine

T immune cells liking T cell,NK cell and LAK cell produce cell factor IL-2 that can integrate with the T lymphocyte surface acceptor IL-2Rαafter activating,which inducing the T cell fast multiplication and producing reject reaction.The cDNA sequence encoding Rong Chang porcine interleukin-2 receptor alpha(PIL-2Rα) was amplified from Rongchang porcine peripheral blood lymphocyte stimulated with Con A by means of using the specific primers based on the reported sequence in GenBank named U₇8317.And sequence identified that the full length of the cloned cDNA was 813bp.Using NCBI website software BLAST to carry out the homology search,finally demonstrated that there were 3 reference sequences(the accession number: NM213835.1 AF036005.1 AF052037.1) the nucleotide homology was very high above 98%.Another pair of primers was designed to clone the gene coding porcine IL-2Rαmature protein which had 774bp.The PET32a(+)/PIL-2Rαwas identified and analyzed by enzymatic digestion and DNA sequcing,and was confirmed.The recombinant strain was transformed into E.coli,BL21,induced by IPTG,SDS-PAGE demonstrated that the fusion protein expressed in form of inclusion body was approximately 45kd.the product of expression account 42.6%of the total bacterium protein.Western-blotting analysis indicated that it was successfully,its dissolubility was identified by SDS-PAGE,Western-blot.The optimal conditions to express mature IL-2Rαwere IPTG 0.2 mmol/L,cultured at 37℃for 4 h.Carting on the immunity test to the white mouse which determined its CD³⁺,CD⁸⁺,CD⁴⁺.The recombinant plasmid group was higher than the pcDNA-3.1(+),the blank control group and does not have any reagent group.The table indicated that IL-2Rαhad certain biological activeness clearly.This experiment clones the Rongchang pig IL-2Rαgene and through expresse the fusion protein after the gene sequence transformation,So this experiment maybe provide research the role of the porcine IL-2Rαin vivo and in vitro,studing the physiological activity of the disease and vaccine and the role of the conditions, Meanwhile local breeds of IL-2Rαin-depth study can be as immune transplant donor reference.