RESUMO
Recombinant Toxoplasma gondii small heat shock protein HSP20, surface antigen SAG1 and dense granule GRA7 were analyzed by IgG-ELISA with serum samples of Toxoplasma infected humans grouped as I (IgG+, IgM+), II (IgG+, IgM-) and III (IgG-, IgM-). rHSP20 reacted against 80% and 62.5% of serum samples from groups I and II, respectively. rSAG1 was recognized by 85% of the samples from group I and 70.8% from group II, whereas rGRA7 was recognized by 85% and 66.6% of the serum samples from groups I and II, respectively. When a combination of two or three recombinant antigens was used, the sensitivity values improved to 85-95% for group I and 87.5-91.7% for group II. All combinations tested produced similar reactivity profiles. None of the recombinant proteins reacted against group III serum samples. In conclusion, we demonstrated that T. gondii HSP20 elicits an important B-cell response during human infection, and could be suitable for the development of serodiagnosis tools.
Assuntos
Proteínas de Choque Térmico HSP20 , Imunoglobulina G/sangue , Complicações Parasitárias na Gravidez/diagnóstico , Proteínas Recombinantes , Toxoplasma/imunologia , Toxoplasmose/diagnóstico , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico HSP20/imunologia , Humanos , Imunoglobulina M/sangue , Gravidez , Complicações Parasitárias na Gravidez/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Toxoplasma/química , Toxoplasmose/imunologiaRESUMO
Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (pâ¯>â¯0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62⯵g/g (±2); biomass from cell suspension cultures 83⯵g/g (±0.2); culture medium from suspensions 71.75â¯mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.
Assuntos
Metaloendopeptidases/antagonistas & inibidores , Planticorpos/farmacologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/farmacologia , Animais , Antivenenos/biossíntese , Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Testes de Neutralização , Planticorpos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
A His-tagged truncated version of Toxoplasma gondii dense granule 4 protein (Gra4(163-345)) was transiently expressed in tobacco leaves. Two genetic constructions were used to accomplish this goal. In one of them, based in a Potato virus X (PVX) amplicon, the sequence encoding His-Gra4(163-345) was placed under control of an additional PVX coat protein subgenomic promoter. In the other, the same sequence was fused to an apoplastic transport signal and placed under the direction of the cauliflower mosaic virus 35S promoter. His-Gra4(163-345) accumulation in agroinfiltrated tobacco leaves was estimated by Western blot analysis using mouse anti-Gra4 antibody and a seropositive human serum. Here, we demonstrated the feasibility of producing a Gra4 antigen using transient expression methods in plants.
Assuntos
Antígenos de Protozoários/biossíntese , Regulação da Expressão Gênica de Plantas , Nicotiana/metabolismo , Proteínas de Protozoários/biossíntese , Toxoplasma/imunologia , Agrobacterium tumefaciens , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Western Blotting , Caulimovirus , Vetores Genéticos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nicotiana/genética , Toxoplasma/genéticaRESUMO
Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 µg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production.