ABSTRACT
Fragments of Clostridium botulinum neurotoxin A (BoNT/A) gene (botA) were expressed in Listeria monocytogenes ATCC10527 to produce the L-chain of the toxin in a soluble form. A shuttle vector pAT19 (EmR) was used to make plasmid pAT-RL containing a botA gene fragment placed under C. botulinum ntnH-gene promoter control. The plasmid also contained a C. botulinum botR/A gene, a positive transcriptional regulator of botA. The cytoplasmic fraction of the L. monocytogenes (pAT-RL) strain was found to contain up to 3 mg/L of a soluble protein of expected size and immunologically positive towards BoNT antibodies. This is the first evidence of heterologous botA gene expression producing a soluble safe derivative of botulinum neurotoxin A needed as a molecular tool for exploratory research in neurosciences as well as a basis for raising protective immunity in humans.
Subject(s)
Botulinum Toxins, Type A/biosynthesis , Clostridium botulinum/genetics , Listeria monocytogenes/genetics , Animals , Antibodies, Bacterial/blood , Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/genetics , Clostridium botulinum/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Electrophoresis, Polyacrylamide Gel , Escherichia coli/chemistry , Escherichia coli/genetics , Listeria monocytogenes/chemistry , Listeria monocytogenes/metabolism , Peptide Fragments/biosynthesis , Peptide Fragments/chemistry , Peptide Fragments/genetics , Plasmids/chemistry , Plasmids/genetics , Polymerase Chain Reaction , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Toxoids/biosynthesis , Transformation, Bacterial/geneticsABSTRACT
Polymerase chain reaction (PCR) for the identification of C.botulinum of type A was developed. As primers, oligonucleotides corresponding to sequences 913 -- 932 and 1852 -- 1871 of the gene of type A botulinic neurotoxin were used. The study revealed that under optimum conditions the positive result of the reaction was registered only when the DNA of C.botulinum strains of type A (11 strains) was used, but not that of C.botulinum strains of other types (11 strains of type B, 5 strains of type C, 2 strains of type D, 6 strains of type E and 1 strain of type G). High sensitivity, specificity and rapidity of PCR open good prospects for its practical use.
Subject(s)
Clostridium botulinum/genetics , Polymerase Chain Reaction/methods , Base Sequence , Botulinum Toxins/genetics , DNA Primers , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Electrophoresis, Agar Gel , Molecular Sequence Data , Sensitivity and SpecificityABSTRACT
Neuraminidase of Corynebacterium-ulcerans was purified by affinity chromatography using immobilized colominic acid preparations. Neuraminidase appears to be a thermolabile protein, molecular mass 70 000 Da. The pH optimum of 5.5 is independent of the substrate used; the optimal temperature is 37 degrees C, the Michaelis constant towards N-acetylneuraminosyllactose is 5.2 X 10(-4) M. Ca2+ and Ba2+ activated the enzyme, but Zn2+, Fe2+, and chelating agent EDTA were inhibitory. In our experiments the enzyme did not hydrolyse the (alpha - 2.6) or (alpha - 2.8) bonds of submaxillary pig mucin and colominic acid, respectively, but it hydrolysed such substrates as fetuin, ovomucin, orosomucoid and horse serum glycoproteins.
Subject(s)
Corynebacterium/enzymology , Neuraminidase/metabolism , Cations, Divalent , Chromatography, Affinity , Edetic Acid/pharmacology , Kinetics , Molecular Weight , Neuraminidase/isolation & purification , Substrate SpecificityABSTRACT
It was studied 203 strains of NAG vibrios and 71 strains of different enterobacteria for the ability to produce neuraminidase. The most frequent neuraminidase producers were found among the strains isolated from humans (99 strain of 131). There was no correlation between neuraminidase production and other properties of the vibrios. The examined strains of the family Enterobacteriaceae did not produce the enzyme.
