Induction of neutralizing antibodies against Tityus serrulatus toxins by immunization with a recombinant nontoxic protein.

An immunogenic nontoxic protein (TsNTxP) was purified from the venom of the scorpion Tityus serrulatus (Ts). This peptide is composed of 63 amino acid residues with a high degree of structural homology with the toxins isolated from Ts. The nucleotide sequence of the gene that encodes TsNTxP was obtained and also showed a high degree of similarity with genes encoding Tityus toxins [Guatimosim, S.C.F., Prado, V.F., Diniz, C.R., Chávez-Olórtegui, C.. Kalapothakis, E., 1999. Molecular cloning and genomic analysis of TsNTxP; an immunogenic protein from Tityus serrulatus scorpion venom. Toxicon 37, 507-517]. In the present study the TsNTxP gene was expressed in E. coli BL21DE3 cells as a fusion protein with maltose-binding protein. The recombinant protein (TsNTxPrec) was purified by affinity chromatography and used as an immunogen in rabbits. The antigenic specificity of anti-TsNTxPrec antibodies was compared by an enzyme-linked immunosorbent assay using TsNTxP, TstFG50 (the fraction of Ts venom that represents most of the toxicity of the crude venom) and the crude venom, to coat microtitration plates. Anti-TsNTxPrec antibodies had a comparable high cross-reactivity for all antigens tested. Concentrations of Ts venom equivalent to 20 LD50 were effectively neutralized by 1 ml of the anti-TsNTxPrec serum. This result provides basic data for the use of such recombinant scorpion protein as an immunogen in the development of antivenoms for clinical use.

Molecular cloning and genomic analysis of TsNTxp: an immunogenic protein from Tityus serrulatus scorpion venom.

A non-toxic protein (TsNTxP) from Tityus serrulatus scorpion venom has been shown to be an efficient immunogen and anti-TsNTxP antibodies recognize and neutralize the effect of Tityus serrulatus venom [Chávez-Olórtegui et al., 1997. Toxicon 35, 213-221]. With the purpose of studying the organization of the gene that code for this protein, we have isolated a full length cDNA clone for TsNTxP from a cDNA expression library using anti-TsNTxP antibodies. The nucleotide sequence of the gene that encodes TsNTxP was also obtained and it reveals the presence of an intron within the signal peptide sequence. The TsNTxP gene showed high degree of similarity with genes encoding toxins from scorpions of the genus Tityrus.

Jejunal absorption of trypsin in rat and guinea pig.

Gut absorption is one of the first requirements for the study of the mechanism of a possible anti-inflammatory action of proteases, such as orally administered trypsin. Porcine trypsin absorption was studied in isolated jejunal loops of rats (female Holtzman and male Wistar) and guinea pigs (males) by open-loop perfusion. Trypsin was dissolved in Tyrode solution and the solution perfused at a rate of 0.5 ml/min, at 37 degrees C. Trypsin activity, total protein, and sodium and potassium concentrations were assayed in the jejunal effluent; the values were unchanged throughout the experiments, which lasted 45 to 120 min. Using a high sensitivity ELISA (i.e. pg/ml), trypsin absorption could be demonstrated by determination of the enzyme in the mesenteric venous blood (samples of 0.5 ml); the enzyme concentration increased with time of perfusion. The linear range-specificity for intact trypsin varied from 1 to 500 ng/well. In this assay polyclonal antibodies prepared against trypsin-TLCK were utilized. Whereas trypsin concentration in the perfused lumen was practically constant at 0.12 mg/ml, the concentration of absorbed trypsin in mesenteric vein blood increased from about 100 ng/ml at time zero to 1.8 micrograms/ml, after 45 min of perfusion. Histological and ultrastructural examination of the jejunal mucosa before and after perfusion revealed that the brush-border, basal membrane, and junctional complexes were fully preserved, thus eliminating the possibility that trypsin might have destroyed the structures, thereby reaching the blood circulation. The present data indicate that micrograms quantities of trypsin were absorbed by the isolated jejunal loop of the rat.

Jejunal absorption of trypsin in rat and guinea pig

Gut absorption is one of the first requirements for the study of the mechanism of a possible anti-inflammatory action of proteases, such as orally administered trypsin. Porcine trypsin absorption was studied in isolated jejunal loops of rats (female Holtzman and male Wistar) and guinea pig (males) by open-loop perfusion. Trypsin was dissolved in Tyrode solution and the solution perfused at a rate of 0.5 ml/min, at 37§C. Trypsin activity, total protein, and sodium and potassium concentrations were assayed in the jejunal effluent; the values were unchanged throughout the experiments, which lasted 45 to 120 min. Using a high sensitivity ELISA (i.e. pg/ml), trypsin absorption could be demonstrated by determination of the enzyme in the mesenteric venous blood (samples of 0.5 ml); the enzyme concentration increased with time of perfusion. The linear range-specificity for intact trypsin varied from 1 to 500 ng/well. In this assay polyclonal antibodies prepared against trypsin-TLCK were utilized. Whereas trypsin concentration in the perfused lumen was practically constant at 0.12 mg/ml, the concentration of absorbed trypsin in mesenteric vein blood increased from about 100 ng/ml at time zero to 1.8 µg/ml, after 45 min of perfusion. Histological and ultrastructural examination of the jejunal mucosa before and after perfusion revealed that the brush-border, basal membrane, and junctional complexes were fully preserved, thus eliminating the possibility that trypsin might have destroyed the structures, thereby reaching the blood circulation. The present data indicate that µg quantities of trypsin were absorbed by the isolated jejunal loop of the rat