(Presented: The Fifth National Conference on Science and Technology, Nov. 10-12, Page no. 184-185)
U.T. Shrestha1, G.S. Sahukhal1, K.B. Tiwari1, A. Singh2 and V.P. Agrawal1
1. Research Laboratory for Biotechnology and Biochemistry (RLABB);
Maitidevi Plaza, Maitidevi, Kathmandu, Nepal
2. Central Department of Microbiology (CDM), Tribhuvan University;
Kirtipur, Kathmandu, Nepal.
ABSTRACT
Bacillus thuringiensis strains were isolated from soil samples collected from Khumbu, Mt. Everest region and characterized by standard microbiological methods. Crystal protein (Delta-endotoxin) was extracted from the crystal protein producing strains (46 from Phereche and 40 from Sagarmatha National Park) from stationary phase culture broth and tested for insect bioassay. The B. thuringiensis strains possess stronger insecticidal activity (~100%) against Dipteron insects (Mosquito) as compared to those of isolates from Kathmandu valley (£60%). Crystal proteins were then further purified and assayed. Its crystal protein had molecular weight about 120-130KD (revealed by SDS-PAGE) and was used to produce polyclonal antibody in New Zealand’s white rabbits. The presence of polyclonal antibody was confirmed by Ouchterlony double diffusion method. Indirect ELISA was optimized against 6-8 mg crystal protein coated per well in microtitre plate. The optimal dilution of the polyclonal antibody was 1000 folds corresponding to OD450 = 0.045 for color observation. Of the total 86 crystal protein producing isolates, crystal proteins from 31 isolates (36.05%) were 25-30% crossreactive, two groups of 6 isolates (6.97%) were 75-80% and 85-90% crossreactive respectively, and 4 isolates (4.65%) were 80- 85% crossreactive with the polyclonal antisera. Only 3 isolates (3.49%) were more than 90% crossreactive. The Discriminatory Index (D) of the Indirect ELISA was 0.92.
Address for Correspondence
Upendra Thapa Shrestha
Research Scientist
Research Laboratory for Biotechnology and Biochemistry (RLABB);
Maitidevi Plaza, Maitidevi, Kathmandu, Nepal
P. O. Box 11537, Sundhara, Kathmandu, Nepal
Mobile: 977-9841431476
Email: upendrats@gmail.com
Website: www.upendrats.blogspot.comto be 0.92.
