Molecular cloning and hyperexpression of a Bt gene, cryIAc, in Escherichia coli DH5 alpha: Production and usage of anti-CryIAc antibody

Abstract

The gene coding for a Lepidoptera-specific insecticidal crystalline (or control) protein (ICP), recognized as cryIAc, from Bacillus thuringiensis subsp. kurstaki HD-73, was cloned into the vector pBluscript II SK-, and then transformed in Escherichia coli DH5 alpha. The clone was named EBtIAc and the chimeric phagemid, as pEBtIAc. Hyperexpression of CrylAc protoxin was observed in the extract of the culture of E. coli harboring pEBtIAc. Crystalline protoxin was purified by differential solubility. It was dissolved in alkaline pH, and exposed to trypsin to be activated. The molecular weights of the pro- and activated toxins on SDS-PAGE were estimated to be ca. 130 kDa and 60 kDa, respectively. The toxicity was tested by force-feeding larvae of gypsi moth (Lymantria dispar) with trypsinized protoxin. Using the batch of biologically active form of the toxin as an immunogen, anti-CryIAc antiserum was raised in a New Zealand white rabbit. Immunoglobulin G was fractionated from the serum by Protein-A sepharose affinity chromatography. Immunoreactivity of the antibody was examined by dot and Western blottings. It has been found that the anti-CryIAc antibody recognized the purified toxin at a level below a nanogram in terms of quantity. Using the antibody some of Bt-coms were able to be differentiated from 'tons of corn kernels which were imported from America as forage crops.