XLIII Annual Meeting of SBBq
th
th
Foz do Iguaçu, PR, Brazil, May 17 to 20 , 2014
Molecular Cloning and Expression of Disintegrins from the Venom of
Bothrops jararaca
David, V.1; Guerrero, T. N.1; Geraldo, R. B.1; Albano, R. M.3; Wemelinger L. S.2;
Zingali, R. B.1;
1
2
Instituto de Bioquímica Médica Leopoldo de Meis, UFRJ, RJ, Brazil;
Departamento de Análises Clínicas e Toxicológicas, UFRJ, RJ, Brazil;
3
Departamento de Bioquímica, UERJ, RJ, Brazil;
Introduction: Snake venoms contain several components, including disintegrins
that are a family of small peptides able to bind to integrin receptors. These
integrins are glycoproteins that play a fundamental role in regulating physiological
and pathophysiological processes, such as in arterial thrombosis. Disintegrins
have been used to develop new drugs some are now used in treatment of human
diseases. Two disintegrins isolated from Bothrops jararaca, in our laboratory,
called jarastatin (JARC) and jararacin (JAST), have the ability to inhibit platelet
aggregation. In this study, we aim to clone the rJARC and rJAST disintegrins from
the venom of Bothrops jararaca using the vector pET-15b, aiming to have enough
amount of peptide to structural studies, once the last vector pET-32a was
unsuccessful. Materials and Methods: The disintegrins were amplified from the
cDNA library using Bothrops jararaca gland. Then, an agarose gel 1.5% was
performed and the DNAs were extracted and digested with restriction enzymes.
The products of these reactions were subcloned into Pet-15b and transformed in
bacteria Escherichia coli BL21. After that, E. coli was coated on agar, containing
ampicillin and chloramphenicol, and incubated at 37°C for 16h. The colonies
obtained were grown in circle growth medium to verify the integrity of the plasmid
by enzymatic digestion and the expression. Results and Discussion:
Amplification of the cDNA was confirmed by the profile of agarose gel, showing
bands corresponding to 243 bp of inserts of disintegrins. After transfection of the
plasmid the bacteria growth was observed as colonies on the plates. In addition,
the integrity of the plasmid was confirmed to rJAST through the agarose profile gel
that showed bands of 243 bp and the expression was successful, while rJARC
showed several random bands. Conclusions: It was possible to clone the rJAST
successfully. New trials in different conditions are required for cloning of rJARC.
Keywords: Disintegrins, haemostasis and thrombosis.
Financial Support: Faperj, CNPq and Capes.
Bibliographic Reference:
WERMELINGER, Luciana S; GERALDO, Reinaldo B; FRATTANI, Flavia S;
RODRIGUES, Carlos R.; JULIANO, Maria A; CASTRO, Helena C; ZINGALI,
Russolina B. Integrin inhibitors from snake venom: Exploring the relationship between
the structure and activity of RGD-peptides. Archives of Biochemistry and
Biophysics. Vol. 482, p. 25–32. 2009.
XLIII Annual Meeting of SBBq
th
th
Foz do Iguaçu, PR, Brazil, May 17 to 20 , 2014
Figure 1: Amplification of the cDNA was confirmed by the profile of agarose gel,
showing bands corresponding to 243 bp of inserts of disintegrins.
Figure 2 and 3: The integrity of the plasmid was confirmed to rJAST through the
agarose profile gel that showed bands of 243 bp and the expression was
successful, while rJARC showed several random bands.