RESUMO
Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.
Assuntos
Toxina da Cólera/genética , Toxina da Cólera/isolamento & purificação , Genes Bacterianos/genética , Histidina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Glândulas Suprarrenais/citologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Linhagem Celular , Toxina da Cólera/biossíntese , Toxina da Cólera/química , Códon/genética , DNA Recombinante/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Histidina/genética , Dados de Sequência Molecular , Subunidades Proteicas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Vibrio cholerae/químicaRESUMO
The antimicrobial susceptibility and the presence of a heat-stable toxin were researched into 100 non-01 Vibrio cholerae strains sent by 7 different health centers to the National Reference Laboratory of Acute Diarrheal Diseases in "Pedro Kourí" Tropical Medicine Institute. The presence of 20% toxigenic non-01 Vibrio cholerae was detected, a figure substantially higher than that reported in other geographic areas, except for endemic areas. This result will make it possible to set epidemiological alert in Cuba because these strains can be infected by CTX phages (element transporting genes that encode for choleric toxin) which will give such strains an epidemic potential similar to that of the etiologic agent of cholera. The identified strains could be studied as possible cholera vaccine candidates.
Assuntos
Toxina da Cólera/isolamento & purificação , Vibrio cholerae/isolamento & purificação , Animais , Técnicas de Tipagem Bacteriana , Pré-Escolar , Toxina da Cólera/análise , Cuba , Fezes/microbiologia , Humanos , Lactente , Camundongos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Sorotipagem , Vibrio cholerae/classificação , Vibrio cholerae/patogenicidadeRESUMO
The antimicrobial susceptibility and the presence of a heat-stable toxin were researched into 100 non-01 Vibrio cholerae strains sent by 7 different health centers to the National Reference Laboratory of Acute Diarrheal Diseases in "Pedro KourÝ" Tropical Medicine Institute. The presence of 20 toxigenic non-01 Vibrio cholerae was detected, a figure substantially higher than that reported in other geographic areas, except for endemic areas. This result will make it possible to set epidemiological alert in Cuba because these strains can be infected by CTX phages (element transporting genes that encode for choleric toxin) which will give such strains an epidemic potential similar to that of the etiologic agent of cholera. The identified strains could be studied as possible cholera vaccine candidates.
Assuntos
Humanos , Animais , Lactente , Pré-Escolar , Camundongos , Toxina da Cólera/isolamento & purificação , Vibrio cholerae , Técnicas de Tipagem Bacteriana , Cuba , Fezes , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Sorotipagem , Toxina da Cólera/análise , Vibrio choleraeRESUMO
Entre febrero de 1992 y 1995 se detectaron 41 casos de gastroenteritis asociada a Vibrio cholerae no O1 en Orán, Salta. La frecuencia de aislamiento fue del 0,9 o/o, entre los casos de diarrea. El 51,2 o/o de los casos correspondió a mayores de 15 años y el 60,9, al sexo masculino. Todos los pacientes tuvieron diarrea, en 24 (58,5 o/o) fue líquida y en 6 (14,5 o/o) con aspecto coleriforme. Diez (24,4 o/o) de los pacientes presentaron vómitos y 12 (29,3 o/o) deshidratación leve o moderada. Seis pacientes pediátricos, desnutridos de 2§ y 3§ grado, que presentaron diarrea de más de una semana de evolución y deshidratación moderada, requirieron hospitalización durante 7 días. Durante el primer brote, en un paciente se aisló simultáneamente V. cholerae no O1 y Shigella flexneri y en el cuarto brote en otro se detectó la asociación V. cholerae no O1 y Salmonella subespecie IV 50: b:-. Una mujer de 72 años falleció durante el segundo brote. El cuadro clínico estuvo caracterizado por diarrea líquida, vómitos, fiebre y deshidratación moderada. De su coprocultivo se recuperó V. cholerae O5 negativo para los siguientes factores de virulencia: toxina de cólera (CT), enterotoxina termoestable, hemolisina (El Tor) y hemaglutinina asociadas a células resistentes a D-manosa y L-fucosa. La caracterización bioquímica de los 41 aislamientos correspondió a V. cholerae con serología negativa para los serogrupos O1 y O139. Ningún aislamiento produjo CT. El 19,5 o/o presentó resistencia a la ampiclina y 4,9 o/o, a trimetoprimasulfametoxazol. La vigilancia activa de las diarreas en Orán, ha demostrado que V. cholerae no O1 no es un agente causal importante de las mismas
Assuntos
Humanos , Masculino , Feminino , Diarreia/epidemiologia , Gastroenterite/epidemiologia , Vibrio cholerae/isolamento & purificação , Vibrio cholerae/patogenicidade , Argentina/epidemiologia , Toxina da Cólera/isolamento & purificaçãoRESUMO
Entre febrero de 1992 y 1995 se detectaron 41 casos de gastroenteritis asociada a Vibrio cholerae no O1 en Orán, Salta. La frecuencia de aislamiento fue del 0,9 o/o, entre los casos de diarrea. El 51,2 o/o de los casos correspondió a mayores de 15 años y el 60,9, al sexo masculino. Todos los pacientes tuvieron diarrea, en 24 (58,5 o/o) fue líquida y en 6 (14,5 o/o) con aspecto coleriforme. Diez (24,4 o/o) de los pacientes presentaron vómitos y 12 (29,3 o/o) deshidratación leve o moderada. Seis pacientes pediátricos, desnutridos de 2º y 3º grado, que presentaron diarrea de más de una semana de evolución y deshidratación moderada, requirieron hospitalización durante 7 días. Durante el primer brote, en un paciente se aisló simultáneamente V. cholerae no O1 y Shigella flexneri y en el cu
Assuntos
Humanos , Masculino , Feminino , Gastroenterite/epidemiologia , Diarreia/epidemiologia , Vibrio cholerae/isolamento & purificação , Vibrio cholerae/patogenicidade , Argentina/epidemiologia , Toxina da Cólera/isolamento & purificaçãoRESUMO
We studied 40 Vibrio cholerae strains: 16 from stool, 16 from sewage and 8 from food. The serotypes were Inaba in 21 strains, 8 Ogawa strains and 11 V. cholerae non-O1. PCR was made with ctx2 and ctx3 primers with 25 cycles of temperature: 1 min at 94 degrees C, 1 min at 60 degrees C and 1 min at 72 degrees C. 24 V. cholerae strains were positive: 18/24 Inaba y 6/24 Ogawa. PCR was negative for 16 strains: 3 Inaba serotype, 2 Ogawa y 11 V. cholerae non-O1. In Vero culture cells 18 strains were cytotonic, 21 cytotoxic and 1 strain was negative. ELISA was positive for 11 strains with PCR positive. The PCR sensitivity was 95.83% compared with culture cells. V. cholerae O1 produced cytotoxic effect on Vero culture cells, maybe related to ACE factor. Colony blot was made with a specific probe labeled with digoxigenin and it could detect 4 Vibrio cholerae toxigenic strains with PCR negative. All V. cholerae Non O1 strains were PCR negative.
Assuntos
Toxina da Cólera/isolamento & purificação , Reação em Cadeia da Polimerase , Células Vero/efeitos dos fármacos , Vibrio cholerae/isolamento & purificação , Animais , Sequência de Bases , Chlorocebus aethiops , Cólera/microbiologia , Toxina da Cólera/genética , Toxina da Cólera/farmacologia , Ensaio de Imunoadsorção Enzimática , Fezes/microbiologia , Microbiologia de Alimentos , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sensibilidade e Especificidade , Sorotipagem , Esgotos , Vibrio cholerae/classificação , Vibrio cholerae/genéticaRESUMO
A cholera toxoid was prepared by iodinating purified cholera toxin having an activity of 25 Limit of blueing (Lb) doses/l ug with 0.8 umol of iodine monochloride per mg toxin, and the residual lesion capacity was tested in mice. The Blueing Dose (BD) test was strongly positive for the native toxin, and co0mpletely abolished in the iodinated toxoid when tested at up to 25 times one Lb dose. The dermal microscopic lesions with intradermal doses of 1 ug virulent toxin presented intense leucocyte infiltration, proteinaceous edema and active hypertemia, whereas none of these effects was observed with the same amount of toxoid. To determine antigenicity, two groups of micereceived toxin or toxoid, 8.5 ug adsorbed to aluminum hydroxide, followed by a booster of 17 ug in saline 21 days later. Measurement of antibodies by ELISA at day 28 indicated that the toxoid was 2.5 times more antigenic than the toxin. These data show iodination converts cholera toxin to an effective toxoid