Human Beta-interferon And Escherichia Coli O157: H7, Surface Antigens Espa Food-grade Lactococcus Lactis Inducible Expression System For Expression And Immune Activity Analysis,

Lactococcus lactis is a Gram-positive,nonpathogenic,noninvasive,nonclonizing and generally regard as safe bacterium.It is mainly used for the production of fermented milk products and daily food industries for a long time.In the last 25 years,impressive progress has been made in the development of genetic engineering tools and the molecular characterization of lactococci.The tools include transformation,the availability of many different vectors,gene intergration,gene knock out,conjugation,and constitutive and regulated gene systems.At present the genome information of at least three strains of L.lactis is elucidated and publicly available.The availability of an easy-to-operate and strictly controlled food-grade expression system-nisin controlled expression system(NICE)has been developed and used for many application.This article is divided into two parts.It is mainly about expression of recombinant human interferon beta 1b and Escherichia coli O157:H7 EspA antigen in recombinant L.lactis strain using food-grade expression system.We aim to in situ delivery of heterologous cytokines and antigens by recombinant strains for preventing and treatment of related diseases.Partâ… secretion of human interferon-beta 1b in Lactococcus lactis for intestinal immunotherapyInflammatory bowel disease(IBD)is caused by excessive and tissue damaging chronic inflammatory responses in the gut wall,including Crohn's disease and ulcerative colitis.The cause of IBD involves genetic,enviromental and immune factors.Human interferon beta was one of the first discovered cytokines.It exerts many biological functions,such as antivirus,anti-proliferation and immuno-regulatory. Many evidences show that IFN-α/βis relevant to the protection against bacterial and parasitic infections.In 2005,Katakura et al.had discovered a protective role of IFN-α/βin a RAG1^-/-murine model of experimental colis,through the activation of a Toll-like receptor 9-dependent signal pathway.Moreover,IFN-βhas also been evaluated in pilot clinical trials in active ulcerative colitis.In this study,we successfully secreted bioactive human IFN-βlb in L.lactis using food-grade expression system.The site-direct mutagenesis was successfully introduced into the recombinant ifn-β1b gene in which 17 Cys was changed to Ser. The PCR product was double digested by restriction enzymes and cloned it into pSec:Nuc vector.In order to increase recombinant protein secretion,a nine-residue propeptide was inserted between usp45 signal peptide and ifn-β1b gene.The two expression plasmids were transferred by electroporation into L.lactis NZ9000 respectively.The recombinant IFN-βwas expression in the recombinant strains and the expression level and biological activity of recombinant proteins were both confirmed.The production of secreted form of IFN-βfrom recombinant strain L. lactis NZ9000/pSec-LEISS-IFNb and pSec-IFNb in culture was about 20μg L^-1and 6.2μg L^-1.Moreover,about 95%of total recombinant protein from the L.lactis NZ9000/pSec-LEISS-IFNb was secreted into the culture medium.The biological activities of recombinant IFN-β1b in both strains were determined to be 1×10⁷ i.u. mg^-1.Since the in situ delivery of recombinant strains has many advantages on low cost,safety and fewer side effects,the use of L.lactis to deliver IFN-βwill provide an alternative or complementary option for the treatment of ulcerative colitis in future.Partâ…¡expression of Escherichia coli O157:H7 espA gene in Lactococcus lactis and oral administration to induce systemic anti-EspA response in miceEnterohemorrhagic Escherichia coli O157:H7(EHEC)is an important zoonotic pathogen of humans,causing severe diarrhea(hemorrhagic colitis)and in a small percentage of cases,haemolytic-uremic syndrome(HUS).Unfortunately,antibiotics increase the risk of HUS,and there are currently no therapies for EHEC human infection other than general supportive measures.EspA,a protein also expressed from the LEE(locus of enterocyte effacement)pathogenity island and essential for A/E lesion formation,is one of the TTSS(Typeâ…¢secretion system)translocator proteins and a major if not the only component of a filamentous structure which extends from the basic needle complex of the secretion apparatus and connects the pathogen to the plasma membrane of the host cell.Many experiments found that EspA protein can cause immune response in vivo.In this study,we successfully expressed the recombinant EspA protein in L.lactis using food-grade expression system.The espA gene was amplified by polymerase chain reaction(PCR)from the genomic DNA of E. coli O157:H7.The espA gene was optimized by lactic acid bacteria bias codons and cloned into pSec:Nuc vector.Western blot analysis demonstrated that the EspA protein was expressed in the L.lactis transformant.The oral administration of the transformants into mice significantly induced the anti-EspA antibody IgG in serum. These results suggest that recombinant L.lactis which expressed EspA can cause specific immune response and may be applicable as an oral vaccine to induce protective immunity against E.coli O157:H7 infection.