CONSTRUCTION OF A RECOMBINANT BACULOVIRUS WITH A NOVEL INSECTICIDE TOXIN LiTx3 OF SPIDER Loxosceles intermedia

SILVESTRE, F.G.(1), CASTRO, C.S. (1), OLORTEGUI, C.C. (1), GOMEZ, M.V. (1), RIBEIRO, B.M.(2), KALAPOTHAKIS, E. (1)

(1)Laboratory of Molecular Biology of the Toxins, Farmacology Department, ICB, UFMG, Belo Horizonte, MG., (2)Laboratory of Eletronic Microscopic, Celular Biology Department, ICC, UnB, Brasília, DF.

Baculoviruses are used as vectors of heterologous gene expression by the introduction of a desired gene in its genome in the place of a nonessential one under control of a strong promoter. Aiming at the recombinant construction of one baculovirus, for the production of a bioinsecticide of high efficiency and specificity for the lizard of the plague of the maize, we have worked in the identification and toxin tests of the Loxosceles intermedia spider with insecticidal activity for insects of agricultural importance. Our group, from the construction of a cDNA library made with mRNA purified from the venom gland of Loxosceles, isolated the LiTx3. The molecular screening of the clone coding LiTx3 was carried out using degenerate probes, and the automatic sequencing of positive clones, allowed the characterization of LiTx3. This toxin presented a hydrophobic signal peptide, a propeptide signal rich in residues of glutamate with arginine as the last residue. The clone also revealed the sequence of the mature toxin and the termination codon beyond the polyadenylation signal. The analysis of the amino acid sequence using the basic local alignment search tool (BLAST) did not show any similarity to described toxins, but the number and the distribution of cisteine is well conserved, following the standard of other toxins. This toxin was subcloned in the vector pSyn XIVVI⁺ X3 and cotransfection using the linearized DNA (vSynVI^- gal) of the AcMNPV virus, directed for the mutated promoter of the polyhedrin. The infection was carried out using the cells Tn5B and the success of the construction was verified by PCR. The expression of LiTx3 in this system was verified by RT-PCR and slot-blot with antibody anti-venom Loxosceles. Preliminary tests with Spodoptera frugiperda had demonstrated insecticidal effectiveness with reduction in the time of life of this plague.

Supported by: CAPES, CNPq, PADCT and FAPEMIG

CORRESPONDENCE TO:

Flávia Galindo Silvestre, Rua Bernardo Guimarães, 1711 apto. 603, Belo Horizonte, MG, CEP: 30140-081, Brasil, Email: flasilvestre@zipmail.com.br