| Listeria monocytogenes(LM) is an important food-borne intracellular pathogen which can cause animal and human serious listeriosis such as meningitis,pyrogenic and septic gastroenteritis,fetal death,premature,births,sepsis,encephalitis,with the mortality about 30%.It is considered as the fourth-largest important food-borne pathogens which was se,cond only to E.coli O157,Salmonella,Shigella by WHO in 2002. Several outbreaks have taken place in the Occident.One side,contamination to food by LM and its serious consequence have gained considerable attention in the worldwide.On the other hand,in the progressive study,we found that LM significantly stimulated a strong response of cellular immunity on CD4~+/CD8~+ T cell but weak in humoral immunity.Generation of an attenuated live vaccine to prevent listeriosis had become an important task.Attenuated LM is a promising vaccine vector for its intracellular parasitism in the treatment of tumor.Because the pathogenesis,of LM was mainly involved in virulence factors that were cooperated with each other,the study of the virulence gene regulation in Listeria monocytogenes takes a important role in the reaserch of listeriosis.In this study,an attenuated recombinant strain LM⊿inlB/actA was obtained by deleting actA virulence genes based on the attenuated⊿inlB LM using homologous recombination method;And the GFP-expression LM strains were constructed,which were used to study the expression of the virulence gene actA regulated by PrfA.The successful construction of the mutant has a very important effect on preventing listeriosis,and establishes a solid foundation to construct vaccine vector to prevent human and animal diseases.Furthermore;it provides the possibilities to elucidate the mechanisms of pathogenesis by LM virulence factors and immune response induced as well as gets a promotion of its clinical applications to improve food hygiene and safety.1.Construction and identification of the attenuated strain LM⊿inlB/actAActA and InlB,encoded by actA and inlB,are essential virulence factors of LM.In this study,we reduced the virulence of a wild type strain LM by deleting actA and plcB. The actA gene was knocked out basing on an attenuated⊿inlBLM strain The flanking DNA fragments of upstream and downstream of actA were amplified and spliced by cloning vector pUC18 and inserted into the shuttle vector pLSV101.Then the recombinant plasmid was introduced into the attenuated⊿inlBLM strain by eleetroporation.Under the pressure of temperature and erythromycin with two rounds of recombination,LM⊿ inlB/actA mutant was obtained by bacteria cloning PCR.2.Use of GFP to study the expression of the virulence gene actA regulated by PrfA in Listeria monocytogenesPrfA is the only regulatory protein identified to date to be necessary for the regulation of the expression of most of the virulence genes in L.monocytogenes.To study the molecular mechanism of the PrfA-dependent virulence genes expression,a new expression vector pLSV16-PactA-gfp was constructed by incorporation of a promoter of virulence gene actA into upstream of a promoterless green fluorescent pr-otein gene gfp, and then electroporated into L.monocytogenes P14(wild type),P14a(PrfA high expression mutant),A42(prfA deletion mutant).The expression level of actA regulated by PrfA was therefore evaluated with the fluorescence intensity of GFP protein.The results showed that the highest fluorescence intensity was observed in P14a,the following was in P14,where the weakest in A42 as well.It suggested that the expression of actA was dependent on PrfA regulation.GFP reporter system could facilitate the research on the regulation of the expression of the PrfA-dependent virulence genes in Listeria monocytogenes.
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