Expression Of Porcine Interleukin-18 In Lactobacillus Casei

The interleukin-18(IL-18)is an important cytokine mainly produced by macrophages and can enhance cell-mediated immunity by upregulation of interferon-y.IL-18 is implicated with anti-microbial,anti-tumor as well as resistance allergic reaction,therefore has the potential as a novel adjuvant of conventional vaccine.The balance between gut microbes and the body's immunity could be effectively enhanced by the Lactobacillus which was one of the first to be widely used probiotic.Lactobacillus which was found to be the little nonpathogenic bacteria so far could tolerate strong acid in gastric juice and bile salts of the upper small intestine,therefore,some Lactobacillus casei strains filtered could be served as a vehicle and deliver exogenous protein.In addition,the Lactobacillus casei strains accounted for a great part of the total microorganism,so Lactobacillus casei serving as an expression vector could obtain a high expression level,compared with other expression vectors through the oral route.In this study,the porcine IL-18 gene would be cloned into Lactobacillus casei with the help of shuttle vector pIAbete8 and recombinant vector pMJ67,so that the recombinant strain could express porcine IL-18 protein.The recombinant strain including porcine IL-18 gene could pose effective double function of immunoenhancement and antibacterial,and could be fed to animals through the oral route,which is rather convenient in clinical application.In this study,the signal peptide(SP)gene of L.brevia was firstly synthesized,which was consisted of 150 bp including Nde ? site in the 5' terminal and the sites in turn Kpn ?,Sma ?,BamH ?,Xba ?,Sal ?,Pst ?,Sph ?,Hind ? and EcoR I in the 3' terminal.The 150 bp fragment was cloned into vector pUCK,which construced vector pUCK-SP.The porcine IL-18 gene spaning 492 bp was obtained from the pMD18-IL-18 vector by PCR amplification,then was inserted Kpn I and EcoR I sites of plasmid pUCK-SP,which finally produced plasmid pUCK-SP-IL-18.The fragment SP-IL-18 from vector pUCK-SP-IL-18 was cloned into Nde I and EcoR I of the integrative vector pMJ67-SP-IL-18,then was transformed into DH5a competent cells,which produced the integrative vector pMJ67-SP-IL-18.The plasmid sequencing confirmed that Lactobacillus integrative vector pMJ67-SP-IL,18 was successfully.constructed.Another pair of primers was designed and synthesed according to Lactobacillus casei lactose operon(LacT)gene sequence(Z80834)published in Genbank,a 298 bp of gene fragment named lactose promoter(plac)was amplified from Lactobacillus casei genomic DNA,the Pst I and Nde I restriction enzyme sites were respectively added in the upstream and downstream.The plac fragment and SP-IL-18 fragment were linked using T4 DNA ligase,and then the plac-SP-IL-18 fragment was amplified by PCR using the plac upstream primer and porcine IL-18 downstream primer.The plac-SP-IL-18 fragment wasligated to the shuttle vector pIAbeta8 and transformed into DH5a competent cells.The plasmid sequencing confirmed that shuttle expression plasmid pIAbeta8-plac-sp-IL-18 was successfully constructed.The integrative vector pMJ67-SP-IL-18 and pIAbeta8-plac-SP-IL-18 shuttle expression plasmid were successfully transformed into Lb.casei CECT5276 by electroporation method respectively,as revealed by the erythromycin and chloramphenicol resistant selection,plasmid PCR amplification and plasmid sequencing identification,and the resulting recombinant Lb.casei CECT5276 which was identified by erythromycin and chloramphenicol resistance selection and PCR product sequencing was successfully constructed.The protein level of IL-18 in the recombinant Lb.casei CECT5276 strain including the integrative vector pMJ67-SP-IL-18 and pIAbeta8-plac-SP-IL-18 shuttle expression plasmid with induction for 16 h by the lactose and 0.8%inducing concentration is highest.SDS-PAGE and Westen-blot analysis showed that the recombinant strain expressed the purpose protein of 19 kDa which was consistent with the theory molecular weight of porcine IL-18,which indicated that the porcine IL-18 was expressed in the recombinant Lb.casei CECT5276.