ABSTRACT
Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is an important pest for Brazilian sugarcane. In the present study, we detected two distinct spots in hemolymph from septic injured larvae (HDs1 and HDs2), which are separated by 2DE gel electrophoresis. Both spots were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis, which revealed the sequence VFGTLGSDDSGLFGK present in both HDs1 and HDs2. This sequence had homology and 80 percent identity with specific Lepidoptera antimicrobial peptides called gloverins. Analyses using the ImageMaster 2D software showed pI 8.94 of the HDs1 spot, which is similar to that described to Hyalophora gloveri gloverin (pI 8.5). Moreover, the 14-kDa molecular mass of the spot HDs1 is compatible to that of gloverins isolated from the hemolymph of Trichoplusia ni, Helicoverpa armigera and H. gloveri. Antimicrobial assays with partially purified fractions containing the HDs1 and HDs2 polypeptides demonstrated activity against Escherichia coli. This is the first report of antimicrobial polypeptides in D. saccharalis, and the identification of these peptides may help in the generation of new strategies to control this pest.
Subject(s)
Animals , Anti-Bacterial Agents/pharmacology , Hemolymph/chemistry , Lepidoptera/chemistry , Peptides/pharmacology , Proteins/analysis , Anti-Bacterial Agents/isolation & purification , Larva/chemistry , Mass Spectrometry , Peptides/isolation & purification , Proteins/metabolism , Sepsis/metabolismABSTRACT
AIM: To construct a recombinant vector containing gloverin and express it in vitro by Rapid Translation System(RTS500). METHODS: The gloverin cDNA was amplified by PCR and inserted into the prokaryotic expression vector pIVEX2.3. The recombinant product was identified by PCR and enzyme digestion. The positive reconstructed expression plasmid pIVEX2.3-G was expressed by RTS500 in vitro. The expressed protein was identified by SDS-PAGE and western blotting. RESULTS: Positive recombinant plasmid pIVEX2.3-G was successfully constructed. Target protein of 13.8 Kda with 6-his tag was detected by SDS-PAGE and western blotting. CONCLUSION: Gloverin polypeptide is successfully expressed in inactive E.coli in vitro.
ABSTRACT
Objective To express anti-bacterial peptide gloverin in prokaryotic system and investigate its bioactivity. Methods E.coli BL21 was used to express gloverin. Western blot was used to identify the production and Tachypleus Amebocyte Lysate (TAL) was used to detect the bioactivity of the production. Results The production was identified as gloverin peptide by Western blotting, and the TAL data indicated that the production could neutralize LPS. Conclusion Anti-bacterial peptide gloverin is successfully expressed in E.coli BL21.