Cloning And Prokaryotic Expression Of RongChang And NeiJiang Porcine Interleukin-8(IL-8)

The cDNA sequence encoding RongChang and NeiJiang porcine interleukin-8 (PIL-8) was cloned from the PBMC stimulated with ConA by means of using the specific primers based on the reported sequence in Genbank(M86923). The cDNA was connected to the pMD18-T plasmid, and cloned the ORF of PIL-8 from pMD18-T-PIL-8, then proved to be IL-8 with enzyme digestion and sequence identification. The full length of the cloned cDNA, was 474bp with an ORF composed of 312 nucleosides, which encodes 104 amino acids. It also showed there was 100% homology between the RongChang and Neijiang PIL-8 and other IL-8 sequence published from the Genbank, which was an identity of 100%, and the indentity of deduced amino acids was 100%. Meanwhile, the hydrophobicity,antigenic index and surface probability analysis of PIL-8 protein were the same as the feature of the PIL-8 protein.By the technology of DNA recombination, the PIL-8 cDNA was cloned into Prokaryotic Expression plasmids PET-32(a)+ vector, and was transformed into Ecoli. The recombinant plasmid was induced to expression through the 1mM concentration of IPTG. The results of SDS-PAGE showed that the expression products were about 31KD and reached their expression peaks about 2 hours after inducing and the expression quatity of fusion protein had 22.7% ratio of total thalline protein, while the E. coli without inducing with IPTG didn't express. The expressed inclusion body IL-8 were washed and the biological activity were detected by the agar diffusion experiment. The results indicated that the recombinant porcine IL-8 proteins have the ability to supply the tendency of porcine peripheral blood cells by the test, and had high biological activity.The RongChang and NeiJiang procine IL-8 was cloned firstly and the IL-8 protein were Prokaryotic expressed in this experiment. With the futher studying of the porcine IL-8 in vivo or in vitro, it could be used in the immunological response. Meanwhile, it could be provided the theory evidence that RongChang and Neijiang procine might be Immunization transplantation donator.