Antibodies immunoreactive with lepidopteran-toxic polypeptides and methods of use

Abstract

Disclosed are novel synthetically-modified B. thuringiensis nucleic acid segments encoding δ-endotoxins having insecticidal activity against lepidopteran insects. Also disclosed are synthetic crystal proteins encoded by these novel nucleic acid sequences. Methods of making and using these genes and proteins are disclosed as well as methods for the recombinant expression, and transformation of suitable host cells. Transformed host cells and transgenic plants expressing the modified endotoxin are also aspects of the invention. Also disclosed are methods for modifying, altering, and mutagenizing specific loop regions between the α helices in domain 1 of these crystal proteins, including Cry1C, to produce genetically-engineered recombinant cry* genes, and the proteins they encode which have improved insecticidal activity. In preferred embodiments, novel Cry1C* amino acid segments and the modified cry1C* nucleic acid sequences which encode them are disclosed.

1 - 40 . (Cancelled).  
     
     
         41 . A purified antibody generated by using a polypeptide comprising SEQ ID NO: 59 or 61 as an immunogen.  
     
     
         42 . (Cancelled)  
     
     
         43 . (Cancelled)  
     
     
         44 . (Cancelled)  
     
     
         45 - 58 . (Cancelled).  
     
     
         59 . A purified antibody generated by using a  Bacillus thuringiensis  Cry1C δ endotoxin polypeptide as an immunogen, wherein: 
 the polypeptide comprises one or more amino acid mutations in the loop region between α helices 4 and 5 of domain 1; and  
 the polypeptide comprises one or more amino acid mutations in the loop region between α helices 6 and 7 of domain 1; and  
 the polypeptide exhibits improved insecticidal activity against insects relative to a native Cry1C δ-endotoxin polypeptide.  
 
     
     
         60 . The antibody of  claim 59 , wherein said polypeptide has an alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue for amino acid 219, and an alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine residue for amino acid 148.  
     
     
         61 . The antibody of  claim 60 , wherein said polypeptide has an alanine, leucine, methionine, glycine, or aspartic acid residues for amino acids 219 and 148.  
     
     
         62 . The antibody of  claim 59 , wherein said antibody is generated by using a polypeptide comprising SEQ ID NO: 59 or 61 as an immunogen.  
     
     
         63 . The antibody of  claim 41  or  59 , operatively attached to a detectable label.  
     
     
         64 . An immunodetection kit comprising, in suitable container means, the antibody according to  claim 41  or  59 , and an immunodetection reagent.  
     
     
         65 . A method for detecting an insecticidal polypeptide in a biological sample comprising contacting a biological sample suspected of containing said insecticidal polypeptide with an antibody in accordance with  claim 41  or  59 , under conditions effective to allow the formation of immunocomplexes, and detecting the immunocomplexes so formed.