Purification Of Ascosphaera Apis Chitinase And Preparation Of Its Polyclonal Antibody

Bee chalkbrood disease, caused by Ascosphaera. apis Olive & Spiltoir, was a fungal infectious disease of honeybee larva. It is one of the main honey disease with great potential effect on apiculture development in China. The present research on chalkbrood are mainly on biological character of the Ascosphaera apis such as the optimal cultivating conditions, prevalence situation, pathology and diagnosis etc. Their pathogenic mechanism is still not clear today. How to control the disease is under exploration. There are certain chitins in the body wall and midgut of insects. Peritrophic membrane is a thin layer membrane in midgut. It is consisted of protein, chitin and other carbonhydrate compounds.And it is also a natural barrier in resistance of insects to fungal pathogens.More and more people pay attention to the role of chitinase that it could damage the peritrophic membrane and disrupt the growth and development of insects in the midgut of insects. Chitinase and its activity are indispensable component and element during intruding honeybee's body wall. Therefore, we made studies on the induction, separation and purification of ND-2 Ascosphaera apis chitinase and on its polyclonal antibody preparation. After a series of treatment such as salt out by 85% (NH4)₂SO₄, centrifugation at 4℃10 000g for 30 min, dialysis, enzyme activity- detection of DNS, SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) and purification, we got a single chitinase with the molecular weight 71.71 kDa, which was then injected into New Zealand rabbits to induce immunoreaction for 45 days until the anti-serum preparation. ELISA (enzyme linked immunosorbent assay) analysis showed that the titer of this polyclonal antibody was 1:256 000.Western blot analysis showed that the antibody had a good specificity to the prepared chitinase protein.