A simple and sensitive assay for chitinolytic activity of the recombinant CHT1 proteins from the hard tick H. longicornis using ethylene glycol chitin

You, Myung-Jo; Fujisaki, Kozo

Korean Journal of Veterinary Research 2003;43(1):145-150.

Myung-Jo You, Kozo Fujisaki

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine

Ethylene glycol chitin을 이용한 진드기 H. longicornis 재조합 CHT1 단백의 키틴분해능 검정 연구

유명조, 고조후지사끼

오비히로축산대학 국립원충성질병연구센터

Abstract

To determine effectively the chitinolytic activity of rCHT1 from the hard tick H. longicornis expressed in baculovirus-mediated Spodoptera frugtperda (Sf) 9 cells, a simple and sensitive assay system was established in solid phase using agarose gel containing ethylene glycol chitin as substrate. The various factors affecting the efficacy of the assay were also investigated. The effects of various temperature, dosages of proteins, pH of media and time courses of reaction were examined to verify the sensitivity of assay for chitinolytic activity of rCHT1 protein. It was found that the optimal reactive conditions were $37^{circ}C$ of temperature, 12 to 15 hours of reactive times, $0.1{mu}g$ of protein concentration and pH 5 to 7 of media. Using the assay system designed, the functional activities of H. longicornis rCHT1l protein could be evaluated simply and sensitively.

Key Words: H. longicornis, recombinant CHT, chitinolytic activity, simple assay, ethylene glycol chitin