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Functional characterization and epitope analysis of a recombinant dermonecrotic protein from Loxosceles intermedia spider.
- Source :
-
Toxicon : official journal of the International Society on Toxinology [Toxicon] 2006 Oct; Vol. 48 (5), pp. 509-19. Date of Electronic Publication: 2006 Jul 06. - Publication Year :
- 2006
-
Abstract
- In the present study the recombinant form (recLiD1) of a dermonecrotic protein present in the Brazilian brown spider Loxosceles intermedia venom was expressed in Escherichia coli cells and purified by reversed-phase HPLC using a C8 Vydac column. About 25.8mg of purified recLiD1 was produced from a litre of bacterial culture. SDS/PAGE and immunoblot analysis of the recombinant protein revealed an apparent molecular weight of 32-35kDa. The later result was confirmed by mass spectrometry (32,758Da). recLiD1 displayed dermonecrotic and platelet aggregation activities which were qualitatively similar to that displayed by the crude L. intermedia venom. However, very low sphingomyelinase D enzymatic activity and complement-dependent haemolytic activities were observed. recLiD1 immunized BALB/c mice developed an antibody response. Anti-recLiD1 antibodies recognized L. intermedia venom in an indirect enzyme-linked immunosorbent assay (ELISA) and cross-reacted with crude venoms from L. intermedia, L. gaucho and L. laeta. An in vivo protection assay carried out 5 weeks after the end of the immunization protocol showed that 75% of the vaccinated mice could resist the challenge by 2.5LD(50) of L. intermedia venom. To characterize epitopes associated with protective antibodies, we prepare sets of immobilized synthetic 15 mer overlapping peptides covering the complete amino acid sequences of the recLiD1. Antibodies revealed one antigenic region in the N-terminal part of the toxin. The amino acid sequence of this epitope was found in several dermonecrotic proteins and some of its residues have been implicated with the active site of the toxin.
- Subjects :
- Animals
Epitopes chemistry
Hemolysis drug effects
Humans
In Vitro Techniques
Mass Spectrometry
Mice
Molecular Weight
Necrosis chemically induced
Necrosis pathology
Phosphoric Diester Hydrolases analysis
Phosphoric Diester Hydrolases chemistry
Phosphoric Diester Hydrolases immunology
Platelet Aggregation drug effects
Rabbits
Recombinant Proteins chemistry
Recombinant Proteins immunology
Serine Endopeptidases chemistry
Serine Endopeptidases immunology
Skin pathology
Spider Venoms chemistry
Spider Venoms immunology
Phosphoric Diester Hydrolases toxicity
Recombinant Proteins toxicity
Serine Endopeptidases toxicity
Skin drug effects
Spider Venoms toxicity
Subjects
Details
- Language :
- English
- ISSN :
- 0041-0101
- Volume :
- 48
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Toxicon : official journal of the International Society on Toxinology
- Publication Type :
- Academic Journal
- Accession number :
- 16934304
- Full Text :
- https://doi.org/10.1016/j.toxicon.2006.06.019