Construction of the recombinant strains of Listeria monocytogenes expressing green fluorescent protein and characterization of their biological properties
Abstract:To evaluate the immune responses induced by transmission of the exogenous antigens through Listeria monocytogenes in the study of prokaryotic transmission model of green fluorescent protein(GEP),the promoter of listeria lysine O(LLO) and the GFP gene gfp were fused together by using the SOEing PCR and then integrated to yzuLM1-2 actA and plcB gene fragments through homologous recombination.In this way,GFP was expressed through the function of up-stream LLO promoter.It was verified by PCR amplification that gfp gene could be successfully integrated to listerial genome.The result in animal experiment showed that the LD50 of the recombinant bacteria in mice was 4.31×108,that was significantly reduced to 10 folds in comparison with that of the attenuated strain yzuLM1-2.The sera of mice immunized with recombinant bacteria showed specific band at 27 kDa in Western blot assay and could elicit production of high level of anti-GFP antibodies as demonstrated by ELISA assay.It is evident that the GFP transmitted by the attenuated L.monocytogeneses can induce strong immunogenicity in mice,thus constituting a solid basis to elucidate the molecular mechanism of pathogenesis and immunity triggered by L.moncytogenes.
[1]Duane ASewell,Zhen Kun Pan,Yvonne Paterson.Listeria-basedHPV-16 E7 vaccines li mit autochthonous tumor growth in atransgenic mouse model for HPV-16 transformed tumors〔J〕.Vaccine,2008,26(41):5322-5327.
[2]Schoen C,Loeffler D I M,Frentzen A,et al.Listeria monocyto-genesas novel carrier systemfor the development of live vaccines〔J〕.International Journal of Medical Microbiology,2008,298(1-2):45-58.
[3]Bruhn K W,Craft N,Miller J.Listeriaas a vaccine vector〔J〕.Microbes and Infection,2007,9(10):1226-1235.
[4]Pizarro-Cerda J,Cossart P.Subversion of cellular functions byListeria monocytogenes〔J〕.Journal of Pathology,2006,208(2):215-223.
[5]Vzquez-Boland JA,Dom nguez-Bernal G.Pathogenicity islandsand virulence evolution inListeria〔J〕.Microbes and Infection,2001,264(2):571-584.
[6]Johnson J,Jinneman K,Stel ma G.Natural atypicalListeria in-nocuastrains withListeria monocytogenespathogenicityislandⅠgenes〔J〕.Appl Environ Microbiol,2004,70(7):4256-4266.
[7]殷月兰,朱国强,耿士忠,等.单核细胞增生李斯特菌actA/plcB缺失株的构建及其生物学特性〔J〕.微生物学报,2008,48(3):299-303.
[8]Ikonomidis G,Portnoy DA,Gerhard W,et al.Influenza-specifici mmunity induced by recombinantListeria monocytogenesvac-cines〔J〕.Vaccine,1997,15(4):433-440.
[9]Humphreys TD,Khanolkar A,Badovinac VP,et al.Generationand maintenance ofListeria-specific CD8+T cell responses inperforin-deficient mice chronically infected with LCMV〔J〕.Vi-rology,2008,370(2):310-322.
[10]Jiang S,Rasmussen RA,Nolan KM,et al.Live attenuatedList-eria monocytogenesexpressing HI V Gag:I mmunogenicity inrhesus monkeys〔J〕.Vaccine,2007,25(42):7470-7479.
[11]Paterson Y,Johnson RS.Progress towards the use ofListeriamonocytogenesas a live bacterial vaccine vector for the deliveryof HI Vantigens〔J〕.Expert Rev Vaccines,2004,3:119-134.
[12]Jiang L,Chunlin KE,Xu J,et al.Listeria monocytogenesmu-tants carrying Newcastle Disease Virus F gene fused toitsactAandplcB:In vitro expression and I mmunogenicity in chickens〔J〕.Acta Biochi mica et Biophysica Sinica,2007,39(1):57-66.
[13]Fortinea N,Trieu-Cuot P,Gaillot O,et al.Opti mization of greenfluorescent protein expression vectors forin vitroandin vivodetection ofListeria monocytogenes〔J〕.Res Microbiol,2000,151(5):353-360.
[14]Si mon BE,Cornell KA,Clark TR,et al.DNA vaccination pro-tects mice against challenge withListeria monocytogenesex-pressing the hepatitis C virus NS3 protein〔J〕.Infect I mmune,2003,71(11):6372-6380.
[15]Joseph B,Przybilla K,Stu hler C,et al.Identification ofListeriamonocytogenesgenes contributing to intracellular replication byexpression profiling and mutant screening〔J〕.Journal of Bacte-riaology,2006,188:556-568.