Study of delta Endotoxin immunocross Reactivity of bacillus Thuringiensis Isolates from Khumbu base Camp of The Everest Region

Abstract

Bacillus thuringiensis strains were isolated from soil samples collected from Khumbu Base Camp of the Everest region and subsequently identified by standard microbiological techniques including colonial characteristics, morphological characteristics and biochemical characteristics. The stationary phase culture broth wastested for insect bioassay and delta-endotoxins (crystal protein) were extracted from the crystal endotoxin producing strains (46 from Phereche and 40 from Sagarmatha NationalPark). The crystal proteins were partially purified in alkaline solution and further purified by Native-PAGE. Among the ten randomly selected isolates, B. thuringiensisS cultureand its purified crystal protein showed the highest insecticidal activity. The endotoxin wasfound to contain five subunits with molecular weight 107KD, 83KD, 71KD, 58KD and40KD as revealed by SDS-PAGE. A pair of New Zealand white rabbits were used to raise polyclonal antisera against purified S endotoxin. The presence of polyclonal antibody was confirmed by Ouchterlony double diffusion method against S 6 vi 1 , S 2a , S 4 , S andP antigens. Indirect ELISA was optimized using 6-8μg of the endotoxin coated microtitreplate per well. The optimal dilution of the polyclonal antibody was found to be 1000 foldscorresponding to OD 10 = 0.045 for colour observation. Of the total 86 endotoxinproducing isolates, 31 (36.05%) corresponding endotoxins were 25-30% crossreactive withthe polyclonal antisera. Similarly, 6 (6.97%) were 75-80% and 85-90% crossreactive each,and 4 (4.65%) were 80-85% crossreactive. Only 3 (3.49%) were more than 90%crossreactive. The Discriminatory Index (D) of the Indirect ELISA was found to be 0.92.The antibody raised against the crystal protein was finally confirmed by Western Blot.