Abstract:

The chitinase gene of Manduca sexta was cloned into the fusion expression vector pET_28a and expressed in E. coli BL21(DE3)host cells. After the expression strain was induced for 6－8 hours by 0.5 mmol/L IPTG, the fusion protein chitinase was expressed and detected in inclusion bodies. After denature and renature procedure in Ni²⁺-NTA affinity chromatography column, soluble chitinase was obtained. The authenticity of in vitro renatured protein was confirmed by Western blot. The inclusion body protein band in SDS-PAGE was excised and the protein was extracted. Then the purified protein was injected into New Zealand rabbits to induce immunoreaction. The induction with two injections lasted for 45 days, and then the anti-serum was prepared. ELISA analysis showed that the titer for this polyclonal antibody was 1∶20 000 Western blot analysis showed that the antibody reacted specifically to the expressed chitinase protein.

Key words: Manduca sexta, chitinase, prokaryotic expression, denature and renature, Western blot, antibody preparation