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Transcriptomic basis for an antiserum against Micrurus corallinus (coral snake) venom.

Leão LI, Ho PL, Junqueira-de-Azevedo Ide L - BMC Genomics (2009)

Bottom Line: Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions.The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens.In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil. lucianaleao@butantan.gov.br

ABSTRACT

Background: Micrurus corallinus (coral snake) is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs) from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization.

Results: A total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx) (24%) and phospholipases A(2) (PLA(2)s) (15%). However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA(2)) and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens.

Conclusion: Besides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA immunization may be a viable alternative. In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.

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Related in: MedlinePlus

Induction of immune response in BALB/c mice immunized with DNA vaccine. Total IgG against the selected antigens (DNA from Atg1, Atg2 or Atg5) were detected through ELISA in plates coated with the respective recombinant protein. Lines with black triangles and black squares indicate, respectively, pre-immune and final sera (two weeks after the last immunization). Each point represents an average of three different measures of the same immunization. Inserts show the detections of the same proteins by western-blot.
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Figure 5: Induction of immune response in BALB/c mice immunized with DNA vaccine. Total IgG against the selected antigens (DNA from Atg1, Atg2 or Atg5) were detected through ELISA in plates coated with the respective recombinant protein. Lines with black triangles and black squares indicate, respectively, pre-immune and final sera (two weeks after the last immunization). Each point represents an average of three different measures of the same immunization. Inserts show the detections of the same proteins by western-blot.

Mentions: In order to determine if the DNA immunizations would be capable of generating an immunological response to the proposed 3FTx candidates, ELISA tests were performed using the anti-sera raised from DNA immunization with Atg1 and Atg2 to recognize the respective recombinant proteins. The results are presented in Figure 5, where it is possible to see a much stronger recognition of the recombinant proteins by the immune sera than by the pre-immune one. The highest titers were obtained for Atg2 (1:10240) and then Atg1 (1:5120). These results indicate that there were differences between the production of antibodies in all three candidates. Besides the ELISA tests, immunodetection by Western blotting was performed (inserts in Figure 5). In this case, the recombinant proteins could also be recognized by their respective sera, Atg1 and Atg2. The two tests indicate that the 3Ftx candidates produced antibody titers with DNA immunization in mice. However, the amplitudes of the responses were quite low, reaching optical density values ranging from 0.25 to 0.5.


Transcriptomic basis for an antiserum against Micrurus corallinus (coral snake) venom.

Leão LI, Ho PL, Junqueira-de-Azevedo Ide L - BMC Genomics (2009)

Induction of immune response in BALB/c mice immunized with DNA vaccine. Total IgG against the selected antigens (DNA from Atg1, Atg2 or Atg5) were detected through ELISA in plates coated with the respective recombinant protein. Lines with black triangles and black squares indicate, respectively, pre-immune and final sera (two weeks after the last immunization). Each point represents an average of three different measures of the same immunization. Inserts show the detections of the same proteins by western-blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662881&req=5

Figure 5: Induction of immune response in BALB/c mice immunized with DNA vaccine. Total IgG against the selected antigens (DNA from Atg1, Atg2 or Atg5) were detected through ELISA in plates coated with the respective recombinant protein. Lines with black triangles and black squares indicate, respectively, pre-immune and final sera (two weeks after the last immunization). Each point represents an average of three different measures of the same immunization. Inserts show the detections of the same proteins by western-blot.
Mentions: In order to determine if the DNA immunizations would be capable of generating an immunological response to the proposed 3FTx candidates, ELISA tests were performed using the anti-sera raised from DNA immunization with Atg1 and Atg2 to recognize the respective recombinant proteins. The results are presented in Figure 5, where it is possible to see a much stronger recognition of the recombinant proteins by the immune sera than by the pre-immune one. The highest titers were obtained for Atg2 (1:10240) and then Atg1 (1:5120). These results indicate that there were differences between the production of antibodies in all three candidates. Besides the ELISA tests, immunodetection by Western blotting was performed (inserts in Figure 5). In this case, the recombinant proteins could also be recognized by their respective sera, Atg1 and Atg2. The two tests indicate that the 3Ftx candidates produced antibody titers with DNA immunization in mice. However, the amplitudes of the responses were quite low, reaching optical density values ranging from 0.25 to 0.5.

Bottom Line: Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions.The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens.In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil. lucianaleao@butantan.gov.br

ABSTRACT

Background: Micrurus corallinus (coral snake) is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs) from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization.

Results: A total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx) (24%) and phospholipases A(2) (PLA(2)s) (15%). However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA(2)) and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens.

Conclusion: Besides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA immunization may be a viable alternative. In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.

Show MeSH
Related in: MedlinePlus