Abstract: 【Objective】 The objective of this paper is to study the function of the enzyme in the process of insect life, enzymatic characterization of chitin deacetylases from Hyphantria cunea and to provide a basis for understanding of its function in the insect life, and to provide new targets for the biological control strategy of H. cunea. 【Method】 The conserved domain of chitin deacetylase 2 sequences from three kinds of lepidoptera insects Bombyx mori, Helicoverpa armigera and Choristoneura fumiferana were analyzed and chitin deacetylase gene-specific primers were designed by homologous alignment. A cDNA clone was identified to contain cDNA sequence coding for chitin deacetylase by PCR amplification. The prokaryotic recombinant expression vector pET30a-HcCDA2 was constructed and cloned in E. coli, the protein expression was induced by IPTG and detected by SDS-PAGE electorphoresis. The recombinant baculovirus expression vector pFastBac-HcCDA1 and pFastBac-HcCDA2 were constructed, and liposome transfection method was used to transfect insect cells Hi5, then the P1, P2 and P3 virus were obtained, respectively. The P3 cells supernatant were collected and western blot analysis was applied to detect the chitin deacetylase 1 (from the previous study) and 2 protein expression. The recombinant proteins were crude purified with ammonium sulfate and then the enzymatic characterizations were studied with the 4’-nitroacetanilide (including the optimum temperature, the optimum pH and the effects of different metal ions on enzyme activity).【Result】 The full-length cDNA clone encoded chitin deacetylase 2 in H. cunea was identified (GenBank accession number: KT781841). The cDNA is 1.6 kb in length. The protein encoded by this cDNA is named HcCDA2 and the protein was shown a band on the SDS-PAGE gel with an apparent molecular weight of 61 kD. The specific antibodies reacting to HcCDA2 were obtained by immunizing rabbits. TheHcCDA1 and HcCDA2 were shown as 80 kD proteins in Hi5 cell line detected by Western blot analysis. The study of enzymatic properties showed that the two enzymes which were secreting expression in insect cells possessed catalytic activity, and the optimum temperature were both 50℃, when the temperature reached 80℃, the HcCDA2 almost lost its activity; HcCDA1 enzymatic reaction appropriate pH ranged from 7.0-9.0, and the optimum pH of HcCDA1 and HcCDA2 were both 8.0. Under the optimum reaction conditions, the enzyme activities of HcCDA2 protein were higher than HcCDA1 protein. Mg²⁺, Zn²⁺, Mn²⁺ and Ca²⁺ showed an inhibition on HcCDA1 and HcCDA2, an increasing Zn²⁺ concentration would lead to the enhancement of inhibitory effects on HcCDA1, but Mg²⁺ showed a trend of increasing first and then decreasing on HcCDA1, as well as an increasing Mg²⁺ or Ca²⁺ concentration showed an inhibition on HcCDA2. Co²⁺ and Fe²⁺ showed activation on HcCDA1 and HcCDA2, Fe²⁺ showed activation stably with increasing concentration, while Co²⁺ showed a trend of increasing first and then decreasing. 【Conclusion】 The gene HcCDA2 was cloned from H. cunea, expressed in E. coli shown as a 61 kD protein, and the HcCDA2 protein-specific polyclonal antibodies were obtained by immunizing rabbits. The active HcCDA1 and HcCDA2 proteins were obtained, and both of them showed a catalytic activity in vitro, the optimum temperature and pH were both 50℃ and 8.0, respectively.

Key words: Hyphantria cunea, chitin deacetylase, cloning and expression, enzymatic characterization