Cloning Of Neijiang And Rongchang Porcine And Prokaryotic Expression Of Interleukin-18 Of Neijiang Porcine

Interleukin-18(IL-18) is a cytokine, playing an important role in regulating and controlling the immune system. It has multiple biological activities, such as inducing other cytokines, especially IFN-γ, mediating cytoxicity of the lymphoid cell, regulating the immune system.In the study, the coding sequences of NJ(Neijiang) and RC(Rongchang) porcine IL-18 genes were amplified by the RT-PCR, with the specific primers, and the PCR template RNA of NJ and RC PBMC after being stimulated by ConA, respectively, then the fragments were inserted into pMD18-T vector, and sequenced. The result of the sequencing indicated that both of the coding sequences of NJ and RC porcine IL-18 genes were 579 bp, encoding 193 aa. Comparative analysis of NJ and RC porcine IL-18 showed that they shared a nucleic acid identity 99.0%, and an amino acid identity 97.9%; compared them with other pigs, they shared a nucleic acid identity 98.6%～99.5%, and an amino acid identity more than97.4%; two amino acid substitutions were present in NJ porcine IL-18. and three in RC porcine, compared with IL-18 of NM₂13997, NJ and RC porcine mature IL-18 had similar physical and chemical properties; and NJ and RC IL-18 showed little codon preference.NJ porcine mature IL-18 gene was amplified by PCR, with the template of the coding sequences of NJ porcine IL-18, then inserted into pET-32a(+), constructing the expression plasmid of NJ porcine mature IL-18. The positive recombinant plasmid was transformed into E. coli BL21(DE3), and was induced by IPTG to express the mature IL-18. A fusion protein about 35 kD and its dissolubility was identified by SDS-PAGE, Western-blot. The optimal conditions to express mature IL-18 were IPTG 0.2 mmol/L, cultured at 30℃for 5 h, the comparative content of recombinant mature IL-18 was 49.6%. The purified recombinant mature IL-18 had certain effective on proliferating PBMC by MTT.The research amplified the whole sequence of NJ and RC porcine IL-18 in China for the first time, constructed the prokaryotic expression system of NJ porcine mature IL-18, realized the effective expression of it, tested the biological activity of it, and offered the fundamental basis to research further on the structure and biological activity of IL-18.