The invention relates to a method for determining the activity of a
trichophyton rubrum
keratinase. The method comprises the following steps: (1), constructing an
artificial skin containing epidermis and
dermis structures: resuspending
human skin fibroblast cells in a collagen liquid, dropwise adding into a
cell culture well plate and culturing; after the cells are completely expanded, dropwise adding a
human keratinocyte suspension to the surface of
collagen gel and continuously culturing; carrying out air-
liquid interface culture when the human keratinocytes completely cover the surface of the
collagen gel; and stopping culturing until a cuticular layer is differentiated into 5-8
layers; (2) carrying out
artificial infection on
trichophyton rubrum; and (3) determining the activity of the
keratinase. The
artificial skin, which is constructed by the method disclosed by the invention, becomes a natural
inducer for production of the
keratinase and has the following advantages that firstly, the
artificial skin contains the dermal layer, the epidermal layer and 5-8 cuticular
layers and the
structure and function are closer to those of the
human skin; secondly,
keratin secreted by the
keratinocyte in the epidermal layer is used as a natural substrate to induce the production of the keratinase; and, thirdly, the keratinase produced by inducing the natural substrate
keratin, is accurate, real and stable in the determination of the activity.