In case of tissue invading nematode, proteolytic enzyme was required at their parasitic life. Proteinases obtained from these parasites(Toxocara cans, Ansalds spp. and Trichinella spiralis) were extracted, isolated and further purified. And then the analysis for activity and inhibitory effect of proteinases was performed by appropriate substrate. Determination of protein as a circulating antigen was done in use of infected animal serum with above parasites, respectively. For above experimental objects, following procedures were performed
First, enzymatic activity was measured in use of azocasein and inhibitory effect of porteinase were studied by various inhibitors. Second, partially purified proteins containing enzymatic activity were obtained by ion exchange chromatography, ultrafiltration and electrophoretic elution. Third role of the partially purified protein as a circulating antigen
The results obtained were as follows:
1. Enzymatic activity of each nematode proteinase was varied according to pH. Optimal pH of Toxocara canis, Ansakis spp. and Trichinella spiralis were pH 6.0, pH 5.5 and pH 6.5, respectively. The optimal molarity of buffer was 0.1 M phosphate buffer. Although little difference between these proteinases was observed, temperature stability was at least maintained at 4 C until 5 days.
2. In case of Ansakis spp. and Toxocara canis, enzymatic activity of theseproteinases was considerably inhibited by Leupeptin and EDTA. For maximum enzymatic activity of above proteinases, it was required that cysteine residue of enzyme should be protected. And it was suggested that metallo type was contained in enzyme active site. Proteinase of Trichinella spiralis contained metallo type also.
3. Although partial purification was performed in Ansakis spp. and Toxocara census, proteins maintaining enzymatic activity were identified as a circulating antigen. From SDS-PAGE and immunoblot, 25 kDa was presented in Ansakis spp. Specific antigen of Toxocara cans was 110 kDa protein fraction. 55 and 42 kDa proteins were reacted with normal serum Trichinella spiralis 60 kDa protein fraction was successfully purified from excretory materials in culture. As a result of immune-reaction with Trichinella spiralis infected serum, highly purified 60 kDa protein was maintained antigenicity until final purification step.
|