Prokaryotic Expression Of Canine IFN-γ And IL-2 And Evaluation Of IL-2 Bioactivity

Two pairs of oligonucletide primers were designed to amplify the genes coding for the IL-2 and IFN-γproteins of canine,respectively. The primers were chosen by the analysis of canine relative sequences available in GenBank. The IL-2 and IFN-γgenes of canine were amplified from RNA extracted directly from canine spleen cells which were stimulated with Concavadin A by RT-PCR and cloned into pMD 18-T vector,respectively. The IFN-γand IL-2 genes of canine were sequenced and sequence analysis indicated that the IFN-γcDNA encompassesd an open reading frame of 426 nucletides,encoding a 141-amino acid protein while the IL-2 gene ORF was 468 bases in length,encoding 155 amino acids.Sequence comparision with the selected sequences from GenBank revealed that the IFN-γgene had a high sequence homology of 99.8% and the encoded protein shared a 99.3% amino acid while the nucletide sequence of IL-2 gene is highly similar to the selected sequence,having a nucletide homology of 99.36% and the encoded protein shared a 98.7% amino acid identity with D30710. The PCR products of pMD 18-T-IFN-γand pMD 18-T-IL-2 were inserted into PJLA605 vector,respectively.The recombinant expression plasmids thus constructed,designated PJLA605-CaIFN-γand PJLA605-CaIL-2,were used to express the two proteins. The induction opportunity and inducing time were optimized for higher expression level. The results showed that when induced for 4 hours in logarithmic anaphase,18.0g and 17.8 of wet bacteria per liter could be obtained and the derivative of CaIFN-γand CaIL-2 were about 27.4% and 29.4% of total protein in the host,respectively. The two engineering bacterial strains were highly expressed. The analysis of protein expression revealed that the expression protein only exist in the inclusion bodies. The amounts of bacteria proteins in the inclusion body can be decreased by washing with NaCl and TritonX-100. The dissolved inclusion body was purified with Sephacry1S-200 and the purify was above 90%.The bioactivities of refolding proteins of IL-2 was detected with MTT method.The results showed that biological activity in stimulating the proliferation lymphblats of IL-2 was 2.01,which indicated that the bioactivities of the protein was obvious.