Abstract：To construct a food-grade secretion expression vector and its use in the expression of reporter protein nuclease(NucA),a fragment,Usp45,containing the coding sequence of promoter,ribosomal binding site,secretion signal and the coding sequence for 11 pre-amino acids was amplified by PCR using the genomic DNA of L. lactis MG1363,and then inserted into the food-grade vector pSH91,thus to construct a secretion expression vector pSQZ. In addition,the coding sequence of NucA(nucA)was also amplified from Staphylococcus aureus chromosome and inserted into vector pSQZ under the control with promoter usp45 to construct a recombinant plasmid pSQZ-nucA. Nuclease plate activity and zymogram assays were to be used to detect the NucA activity in the secretion expression of L. lactis. It was demonstrated that the secretion expression vector system L.lactis MBP71/pSQZ-nucA was successively constructed through the processes mentioned above and the quantity of NucA secreted into cultural supernatants was greater that that in bacterial lysates. Because all the fragments used to construct the vector pSQZ were obtained from food-grade bacteria,the vector pSQZ can be regarded as a food-grade secretion expression vector. These data can be used as the foundation for the further study on the secretion expression of the protective antigen in the oral vaccine.
〔1〕Ravn P,Arnau J,Madsen SM,et al.The development of TnNuc andits use for the isolation of novel secretion signals in Lactococcus lactis〔J〕.Gene,2000,242(1-2):347-356.
〔2〕Poquet I,Ehrlich SD,Gruss A.An export-specific reporter de-signed for gram-positive bacteria:application toLactococcuslac-tis〔J〕.J Bacteriol,1998,180(7):1904-1912.
〔3〕Maguin E,Prevost H,Ehrlich SD,et al.Efficient insertional mutagenesis in lactococci and other gram-positive bacteria〔J〕.J Bacteriol,1996,178(3):931-935.
〔4〕Bolotin A,Wincker P,Mauger S,et al.The complete genome sequence of the lactic acid bacteriumLactococcuslactis ssp.lac-tisIL1403〔J〕.Genome Res,2001,11(5):731-753.
〔5〕Bermudez-Humaran LG,Langella P,Commissaire J,et al.Con-trolledintra-or extracellular production of staphylococcal nucle-ase and ovine omega interferoninLactococcus lactis〔J〕.FEMS Microbiol Lett,2003,224(2):307-313.
〔6〕Dieye Y,Usai S,Clier F,et al.Design of a protein-targeting systemfor lactic acid bacteria〔J〕.J Bacteriol,2001,183(14):4157-4166.
〔7〕Schotte L,L Steidler,J Vandekerckhove,et al.Secretion of bio-logically active murine interleukin-10byLactococcus lactis〔J〕.Enzyme Microb Technol,2000,27:761-765.
〔8〕Slos P,P Dutot,J Reymund,et al.Production of cholera toxin B subunit inLactobacillus〔J〕.FEMS Microbiol Lett,1998,169:29-36.
〔9〕Pedersen M B,Jensen P R,Janzen T,et al.Bacteriophage Re-sistance of a△thyA Mutant ofLactococcus lactisblocked in DNA replication〔J〕.Appl Environ Microbiol,2002,68:3010-3023.
〔10〕孙强正,熊衍文,李振军,等.乳酸乳球菌食品级载体的构建及Mn-Sod基因的克隆和表达〔J〕.中国人兽共患病学报,2006,22:498-501.
〔11〕Lachica RVF,Genigeorgis C,Hoeprich PD.Metachromatic agar-diffusion methods for detecting staphylococcal nuclease ac-tivity〔J〕.Appl Microbiol,1971,21:585-587.
〔12〕Holo H,Nes I F.High-frequency transformation,by electro-poration,ofLactococcuslactis subsp.cremorisgrown with gly-cine in osmotically stabilized media〔J〕.Appl Environ Microbi-ol,1989,55:3119-3123.
〔13〕Le Loir Y,Gruss A,Ehrlich SD,et al.Anine-residue synthet-ic propeptide enhances secretion efficiency of heterologous pro-teins inLactococcus lactis〔J〕.J Bacteriol,1998,180:1895-1903.
〔14〕Liebl W,Sinskey A J,Schleifer K H.Expression,secretion,and processing of staphylococcal nuclease byCorynebacterium glutamicum〔J〕.J Bacteriol,1992,174:1854-1861.
〔15〕Wells J M,Wilson PW,Norton PM,et al.Lactococcus lactis:high-level expression of tetanus toxin fragment C and protec-tion against lethal challenge〔J〕.Mol Microbiol,1993,8:1155-1162.
〔16〕Norton PM,Le Page RW,Wells J M.Progress in the develop-ment ofLactococcuslactisas a recombinant mucosal vaccine de-livery system〔J〕.Folia Microbiol(Praha),1995,40:225-230.
〔17〕Ribeiro LA,Azevedo V,Le Loir Y,et al.Production and tar-geting of theBrucella abortusantigen L7/L12inLactococcus lactis:a first steptowards food-gradelive vaccines against Bru-cellosis〔J〕.Appl Environ Microbiol,2002,68:910-916.
〔18〕Leenhouts KJ,Tolner B,Bron S,et al.Nucleotide sequence and characterization of the broad-host-rangelactococcal plasmid pWVO1〔J〕.Plasmid,1991,26:55-66.
〔19〕Le Loir Y,Nouaille S,Commissaire J,et al.Signal peptide and propeptide opti mization for heterologous protein secretion in Lactococcus lactis〔J〕.Appl Environ Microbiol,2001,67:4119-4129.
