Abstract: 【Objective】 Haemonchus contortus is one of the major gastrointestinal nematodes infecting millions of ruminants, the disease (haemonchosis) was nationally epidemic in China. In order to analyze biochemical properties of Hc-DAF-22 in Haemonchus contortus, the enzyme activity of renatured Hc-DAF-22 was measured in this study. By the results of enzyme activity assay, the role of Hc-DAF-22 protein is understood in peroxisomal β-oxidation. 【Method】 First, the full-length of open reading frame of Hc-daf-22 was amplified by PCR from total cDNA of adult H. contortus ZJ Strain （GenBank：HQ738470.1）. A recombinant pET-22b-Hc-daf-22 was constructed and transformed into E. coli BL21 strain; and its prokaryotic expression was induced for four hours by IPTG (0.1 mmol·L^-1). Then bacterial liquid were centrifuged and cells were resuspended in PBS solution (50 mmol·L^-1). After verification by SDS-PAGE and Western Blot, the prokaryotic Hc-DAF-22 was purified by Ni-chelating affinity chromatography and concentrated by ultrafiltration tube. The concentration of protein was measured according to protein concentration assay kit. Natural state of Acetoacetyl CoA can occur keto - enol tautomerase then enol compound were formed. Using this property, taking acetoacetyl coenzyme A as the substrate, the enzyme activity of renatured Hc-DAF-22 was measured in the reaction system (50 mmol·L^-1 Tris-Cl pH 8.1, 20 mmol·L^-1 MgCl₂, 60 μmol·L^-1 CoA, 10 μmol·L^-1 AcAc-CoA, 0.1 μg protein). Different temperatures and pH were set in order to determine the optimum reaction conditions. 【Result】 The results showed that Hc-daf-22 gene was successfully cloned and gene similarity was 99.9% compared with H. contortus ZJ strain. Recombinant Hc-daf-22 could be expressed in E. coli BL21 and it could be detected both in the lysate supernate and precipitates, and molecular weight of the fusion protein was about 59 kD. The enzyme assay indicated that it could catalyze the substrate acetoacetyl coenzyme A, suggesting the activity of thiolase. And the optimal pH of this reaction was 8 and the optimal temperature was 37ºC. Under the optimal reaction conditions, the Michaelis constant Km and the maximum reaction velocity Vmax were 33.765 μmol·L^-1 and 1 784 nmol·L^-1·min^-1, respectively. 【Conclusion】 H. contortus DAF-22 protein is a key enzyme of peroxisome β-oxidation, this experiment is successfully determined of Hc-DAF-22 protein activity in vitro activity test. It proved that Hc-DAF-22 have certain thiolase activity, but is low compared with the Caenorhabditis elegans homolog protein.

Key words: Haemonchus contortus, Hc-daf-22, prokaryotic expression, enzyme activity assay, β-oxidation