To begin, take a polyethylene plastic film 16 centimeters long, 12 centimeters wide, and 20 micrometers thick. Use a glass grinding tool to press out 30 semi-circular protrusions in a standard 96-well PCR layout. Expose both sides of the pressed polyethylene plastic film to UV light for one hour to disinfect it.
Add a small amount of 10%polyvinyl alcohol to the semi-circular surface. Next, place about 3, 000 anesthetized female trichogramma dendrolimi wasps into a collection box. Place the convex side of the film egg card towards the collection box and secure the edges with a rubber band.
Now add four microliters of amino-acid solution to each protrusion. Then cover it with another piece of polyethylene plastic film. Use a rubber band to tightly cover the collection box with two sheets of plastic.
Allow the wasps to parasitize freely for four to eight hours. Provide 10%sucrose water through wet cotton during parasitization. Next, obtain the parasitized amino-acid solution from the inner protrusion of the artificial egg card and transfer it to the cap of 1.45 milliliter tubes.
Cover the tube cap with a 10-micrometer nylon net, having a diameter of 25 millimeters. Then fasten the nylon net in the tube lightly. Centrifuge the upper right tube at 1, 360 g for 10 seconds.
Collect the filtered solution containing the venom. Store the venom at minus 80 degrees Celsius for further analyses. BCA analysis of the venom samples showed that the concentration of venom protein ranged from 0.35 to 0.46 micrograms per microliter.
The negative control of amino-acid solution only had a protein concentration of 0.03 to 0.05 micrograms per microliter. SDS-PAGE analysis of the venom showed a protein range spanning from under 10 kilodaltons to over 130 kilodaltons in size.
