Expression, Purification Of BmK IT And Chitinase And Preparation Of Their Polyclonal Antibodies

Scorpion neurotoxins are very useful models for studying protein structure-function relationship. They have been used as valuable probes for the elucidation of structure and function of ion channels. All kinds of insect-selective scorpion neurotoxins display no or less toxicity toward mammals but are specifically active toward insects. Therefore, they are widely used for studying biological pesticides and anti-insect transgenic plants. Chitin is a major component of insect cuticle and gut cell wall. Insects secrete chitinase to degrade chitin when moulting. If chitinase gene is transferred into and constitutively expressed in plants, it will prevent the normal development of insects that feed on the transgenic plants. In this study, a scorpion insect-selective neurotoxin and an insect chitinase were expressed in procaryotic expression systems and purified, respectively. Polyclonal antibodies were prepared using the purified proteins, which made it possible to detect the expression of exotic scorpion neurotoxin and chitinase in transgenic plants. At the same time, preparation of polyclonal antibodies provides an efficient approach for evaluating and selecting transgenic plants. By now, we have obtained a series of progress as following:An insect excitatory toxin from Buthus martensii Karsch (BmK IT) was cloned into an expression vector pTWIN1. Fusion protein of CBD-intein-BmK IT was obtained in the soluble form. The protein was purified by two anion-exchange chromatography columns and one gel chromatography column. Bioassays were carried out to verify the toxicity of this recombinant toxin. The results indicated that the expressed recombinant BmK IT has biological activity.BmK IT was cloned into the second expression vector pET-28a and expressed as inclusion bodies in Escherichia coli BL21 (DE3) host cells. The authenticity of in vitro expressed protein was confirmed by Western blot. The...