Secretory Expression Of Porcine Interferon- Alpha In Lactobacillus Casei

As an effective anti-viral cytokines, the porcine interferon-alpha (IFN-a) which is one of the pig important early defense systems against pathogenic invasion can quickly interfere with virus replication and effectively prevent the occurrence of infectious diseases. Currently, the recombinant porcine IFN-a which was produced by engineering technology can be available in commerce, and can effectively prevent swine infectious viral diseases including Porcine epidemic diarrhea (PED), classic swine fever (CSF), Porcine Reproductive and Respiratory Syndrome (PRRS) and transmissible gastroenteritis (TGE), etc in practice. The Lactobacillus existed in the gut of humans and animals was a recognized beneficial microorganism and had natural antibacterial effect. Lactobacillus which was found to be the only 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 serve 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 IFN-a gene would be cloned into Lactobacillus Casei with the help of casset vector pIA bete8 and recombinant vector pMJ67, so that the recombinant strain could express porcine IFN-a protein. The recombinant strain including porcine IFN-a gene could pose effective double function of antiviral 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 I site in the 5’terminal and the sites in turn Kpn Ⅰ, Sma Ⅰ, BamH Ⅰ, Xba Ⅰ, Sal Ⅰ, Pst Ⅰ, Sph Ⅰ, Hind Ⅲ and EcoR I in the 3’ terminal. The 150 bp fragment was cloned into vector pUCK, which construced vector pUCK-SP. The porcine IFN-agene spaning 501 bp was obtained from the pMD18-IFN-a vector by PCR amplification, then was inserted Kpn I and EcoR I sites of vector pUCK-SP, which finally produced vector pUCK-SP-IFN-a. The fragment SP-IFN-a from vector pUCK-SP-IFN-a was cloned into Nde I and EcoR I of the recombinant vector pMJ67-SP-IFN-a, then was transformed into DH5a competent cells, which produced the recombinant vector pMJ67-SP-IFN-a.The plasmid sequencing confirmed that Lactobacillus integrated expression vector pMJ67-SP-IFN-a was successfully constructed. According to Lb.casei lactose operon (LacT) gene sequence (Z80834) published in Genbank, a 298 bp of gene fragment named lactose promoter (plac) was amplified from Lb.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-IFN-a fragment were linked using T4 DNA ligase, and then the plac-SP-IFN-a fragment was amplified by PCR using the plac upstream primer and porcine IFN-a downstream primer.The plac-SP-IFN-a fragment was ligated to the shuttle vector pIAbeta8 and transformed into DH5a competent cells. The plasmid sequencing confirmed Lactobacillus integrated expression plasmid named pIAbeta8-plac-sp-IFN-a was successfully constructed.Through electrictransformation method, the pIAbeta8-plac-SP-IFN-a expression vector was successfully transformed into Lb. casei CECT5276, by the chloramphenicol resistant filter, plasmid PCR identification and plasmid sequencing identification, which proved the Lb. casei CECT5276 cintaining expression plasmid replication type pIAbeta8-plac-SP-IFN-a.Using electrictransformation method, the recombinant vector pMJ67-SP-IFN-a was successfully transformed into Lb. casei CECT5276, through erythromycin resistance screening, genomic DNA PCR product sequencing identification. The mRNA level of IFN-a in the recombinant strain with induction for 16 h by the lactose is highest. SDS-PAGE analysis showed that the recombinant strain induced by the lactose for 16 h expressed the purpose protein of 19 kDa which was consistent with the theory molecular weight of porcine IFN-a, which indicated that the porcine IFN-a was expressed in the recombinant Lb. casei CECT5276.