Clonging And Expression Of Interleukin-2 And Interferon-γ Genes Of Guangxi Bama Mini Pig

The interleukin-2 cDNA and interferon- γ of Bama Mini Pig was cloned by RT-PCR from total RNA extracted from blood lymphocytes, which were cultured in RPMI 1640 medium and stimulated with lOug/ml ConA in vitro for 15 hours. The interleukin-2 and interferon- y cDNA of Bama Mini pig were then cloned into T vector sequenced. The length of the IL-2 was 503 bp (ORF was 465 bp), encoding a peptide of 154 amino acids whose molecular weight is 17.4 KDa and pl is 5.38. The nucleotide acid length of the interferon- y was 556 bp (ORF was 501 bp), encoding a peptide of 166 amino acids whose molecular weight is 19.4 KDa and pl is 9.52. The sequences of Bama Mini pig interleukin-2 and IFN-γ gene from lymphocyte are identical to the interleukin-2 genes and interferon- y respectively previously cloned from spleen cell and fibroblast cell of other porcines.Two pairs of primers were designed to sub-clone the two genes coding interlukin-2 mature protein (namely mIL-2) and interferon- y mature protein(namely mIFN). In order to detect the expression of fusion protein by assaying the HA Tag, another two pairs of primers including HA-Tag sequences were designed to sub-clone the mature interleukin-2 fused with HA-Tag(namelyHA-mIL-2) and interferon- Y mature protein fused with HA-Tag(namely HA-IFN-γ ).And the sub-clone mIL-2, IFN-γ, HA-mIL-2 and HA-IFN-γ were inserted to expression vector PET respectively to construct the expression plasmids PET-mIL-2, PET-mIFN, PET-HA-mIL-2 and PET-HA-IFN-γ .The recombinant plasmids were digested by BamHI and HindIII to identify whether the aim fragments were inserted into the plasmid in correct orientation. The recombinants were then transformed into E.coli BL21 competent cells induced by IPTG . Protein expression of the recombinants were assayed by SDS-PAGE. A new protein band was found in SDS-PAGE with molecular mass of about 34.KDa, which is consisted of a 14 Kda protein of the mIL-2 or HA-mIL-2 gene and Trx (20 KDa). And a new protein band was found with molecular mass of about 36.Kda, which is consisted of a 16 Kda protein of the mIFN-γ or HA-mIFN-γ gene and Trx (20 KDa). It indicates that the porcine IL-2 and IFN-γ gene were correctly transcribed and translated in the transformed bacteria, which could be employed in the detection of content and the bioactivity analysis of the porcine IL-2 and IFN- Y in vitro and in vivo. These results proved that the porcine IL-2 gene and IFN- Y and their recombinant proteins could be further applied to develop effective immunoadjuvants to enhance the immunity of animals against infectious diseases.