Polyclonal antibody against Manduca sexta chitinase and detection of chitinase expressed in transgenic cotton.

ABSTRACT

The chitinase gene of Manduca sexta was cloned into the expression vector, pET-28a, and expressed in Escherichia coli BL21 (DE3) host cells. The protein product was expressed in inclusion bodies. After denaturation and renaturation procedures using a Ni2+-NTA affinity chromatography column, soluble chitinase was obtained. The authenticity of the renatured protein was confirmed by Western blotting. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed chitinase and was used to quantify its presence in transgenic cotton being developed to resist attack by various insects.