Electrophoretic Mobility Shift Assay By Argr Repressor From Corynebacterium Crenatum And Construction Of Expression Vector Carrying ActA, InlB,P60from Listeria Monocytogenes

L-Arginine (L-Arg), as a semi-essential amino acid, has numerous applications in food flavor and pharmaceuticals because it is a substantial intermediate in human body's metabolism. Corynebacterium crenatum AS1.542is a Gram-positive bacteria isolated from soil by the scientist in our country and its mutant strains were widely used in producing the kinds of amino acids by the direct fermentation method.ArgR protein is an important regulator in Corynebacterium crenatum. In this experiment,we constructed recombinant strain BL21(DE3)/pET22b-argR. The soluble ArgR expression was induced by IPTG at33℃for8hr. Anti-ArgR polyclonal antibodies were obtained by immuning mice and the titer of anti-ArgR antibodies achived1:160000by the indirect ELISA analysis. Western blot analysis revealed that the antibody could react specifically with the expressed recombinant protein and that the purified ArgR was stable in the form of polymer mediated by L-arginineThe genes involving the arginine biosynthetic pathway in Corynebacterium crenatum, including argC, argJ, argB, argG, argF and carAB were probalbly aregulated by ArgR protein. Electrophoretic mobility shift assay was used to validate the binding action of ArgR and the operator in upstream of six genes, respectively. The results were shown that the ArgR protein could bind efficiently with argB, argF and carAB operator, respectively and bind strong with carAB operator than othersListeria monocytogenes is a bacterial pathogen that causes morbidity and mortality in humans and livestock and it is one of the important routine monitor pathogen in our country. The full lengths of actA,inlB and p60, which encode three specific membrane proteins, respectively, were amplified using the genomic DNA of LM as the template and then cloned into the prokaryotic expression vector pET22b(+). The resulting pET22b(+)-actA,pET22b(+)-inlB and pET22b(+)-p60vectors were transformed into E.coli BL21(DE3). This work will provide the corresponding preparation for the high expression of specific membrane protein antigen of LM.