Isolation, Purification Of Antimicrobial Materials And Cloning, Analysis Of PGRP-S Gene From Protaetia Brevitarsis Larva

Gene-encoded antimicrobial peptides (AMPs), originally characterized in insects, are now recognized as a widely deployed mechanism of biochemical defense against potential pathogens. Although exceptions exist, AMPs characteristically are 100 amino acids or less in chain length, cationic at neutral pH, and have broad-spectrum antibiotic activity against a wide range of microbes and certain viruses at low micromolar concentrations. AMPs have been isolated from all phyla examined, and their expression and release is observed in diverse host defense.Insects are the largest biological populations, which constitute about 75% of all animal species and have colonized nearly every terrestrial habitat. Insects are the biological treasure with great potentials as well as important natural resources with ecological, research, economic and cultural value. A variety of bio-active substances from insect haemolymph, which play an even important role in the host defense of inscet, have attracted significant attention of the researchers all over the world.So far, more and more proteins and polypeptides of all kinds have been isolated and characterized. It is said that there were nearly characterized species of inscet. Our country is rich in insects resource, which approximately takes up 10% of the world. So our country has great potentials in the field of bioactive materials exploration.Two antibacterial materials have been purified from Protaetia brevitarsis larvae haemolymph by Sephadex G-50 gel filtration and two-step reverse phase high-performance liquid chromatography(RP-HPLC).The molecular weights were 2254.0459 and 2262.4438 Da respectively,determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).They inhibited the growth of Bacillus subttilis,which indicated that the two antibacterial materials should play an important role in preventing bacterial infection in P. brevitarsis.Fat body cDNA library of P. brevitarsis larva was constructed. 1×10~6 independent clones were got in the fat body cDNA library.This the first fat body cDNA library of P. brevitarsis larva, which can improve the structure and function research of antibacterial peptide from P.brevitarsis.Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and plays a crucial role in the innate immune responses as a pattern recognition receptor (PRR). By searching the cDNA sequences of insect peptidoglycan recognition proteins on Genbank, we designed special primers for obtainting PGRP from the ds cDNA of P.brevitarsis fat body via PCR method. By sequencing of independent clones, a 623 bp conserved cDNA fragment of a short type PGRP was obtained from P.brevitarsis larva fat body. The deduced amino acid sequences of open reading frames are composed of 174 amino acid residues with an estimated molecular mass of 19.30 kDa and predicted isoelectric point of 8.37, which share 98% homology with PGRP-1 from Holotrichia diomphalia.This is the first time got the PGRP-S from fat body of P.brevitarsis larva.