The cloning of pig interleukin 18 mature protein gene and its expression in Lactic acid bacteria

Objective To clone porcine interleukin 18 mature protein gene and express it in Lactobacillus plantarum NC8(Lb.plantarum NC8).Methods The porcine interleukin 18 mature protein gene(pIL-18) was amplified from its spleen cells by RT-polymerase chain reaction(PCR) method,then subcloned to pMD18-T and sequenced.The positive gene fragment was subcloned to lactic acid bacteria expression vectors PSIP-409 to construct recombinant expression vector PSIP-409-IL-18 and identified by enzyme digestion and PCR.The recombinant expression vector PSIP-409-IL-18 was transformed to Lb.plantarum NC8 through electric perforation techniques.After the recombinant strains were induced to express,the expression product was analysed by SDS-PAGE and Western-blot.Results Sequencing results showed that the pIL-18 mature protein gene contained 579 bp of bases and coded 193 nucleotides.The recombinant expression vector pSIP-409-IL-18 was proved to have been successfully constructed by enzyme digestion and PCR.SDS-PAGE and Western-blot analysis indicated that 18 kD fusion protein was expressed by these recombinant strains and it can react with the IL-18 polyclonal antibody of mouse anti-porcine.Conclusion The pIL-18 mature protein gene was amplified.And the pIL-18 recombinant Lactic acid bacteria with boligical activity was successfully constructed,which lay the foundation for the development of IL-18 recombinant Lactobacillus.