Differential Analysis Of Proteins In Bombyx Batryticatus Before And After Processing

Bombyx batryticatus is dried fungus-infected complex, which is dead and stiffened due to a Beauveria bassiana infection. As a traditional Chinese medicinal material, it has been utilized for thousands of years. Bombyx batryticatus possesses muti-pharmacologic effects including anti convulsion, anti epilepsy, anti coagulation, etc. So it is widely used clinically to treat Convulsions, epilepsy, convulsions, and so on. There are two kinds of medicine materical crude slices of Bombyx Batryticatus, including raw product and its processed products. Because the raw one has strong toxicity, the processed product is often used in clinical application. After processing, toxicity is greatly weakened. Small molecules are studied on the toxicity analysis as always but there has been no conclusion until now. However, proteins as the main component in Bombyx Batryticatus have not been investigated. This thesis focuses on the analysis of the changes of proteins in Bombyx Batryticatus before and after processing and clearly gets the differential proteins. It provides the basis of the detoxification of Bombyx Batryticatus processing. The main parts of study are as follows:1. Bombyx Batryticatus are prepared into processed products. Optimizing the methods of extracting proteins in Bombyx Batryticatus and using water as solvent and 1:50 solid-liquid ratio, ultrasonic for 50 mins as the best extraction method. Determining the protein content of Bombyx Batryticatus included the raw one, homemade processed products and the commercial processed products by using optimal extraction method. The protein content of the raw one is higher than the processed one and homemade processed products have slightly higher protein content than the commercial processed products.2. Hydrolysis of Bombyx Batryticatus using acid hydrolysis and content analysis of amino acid using an amino acid analyzer. It is found that the component of amino acid and content trend are same between in the raw one and the processed one.The amino acid content of the raw one is higher than the processed one but not methionine.3. Using polyacrylamide gel electrophoresis to analyze protein of Bombyx Batryticatus and comparing different concentration of separation gel and voltage on the effect of protein separation. Proteins in both the raw one and processed one are analyzed. It is obviously to see that the numbers of protein bands of raw one are more than processed one that shows rarely protein band. There are no visual protein bands both in homemade processed products and commercial processed products.4. Analysis of the protein differences between the raw one and processed one by using two dimensional gel electrophoresis. It is found that 15% separation gel, silver staining and saturated ammonium sulfate precipitation make the 2-DE spectrum results clearer after comparing the effects of different separation gel concentration, dyeing methods and before and after protein purification. Also by comparing the two-dimension profile between the raw one and processed one, the results show that the spots of the raw one are much more than the processed. But afer the purification, there are several spots that are similar to the raw one.5. Proteins are digested with trypsin after being separated by SDS-PAGE and 2-DE, respectively. Peptides obtained are further analysed by MALDI-TOF-MS. The corresponding peptide fragment fingerprinting is imported to the Mascot database to match with existing proteins. There is only one protein identified with high confidence digested from protein bands. But it is difficult to get credibility proteins as results for protein spots.6. The samples above are then analysed by Nano-LC-ESI LTQ-Orbitrap MS/MS. Using PEAKS software to search through the three databases, the proteins with high confidence and differential proteins are obtained. There are 30 proteins identified with high confidence digested from protein bands and 40 proteins identified with high confidence digested from protein spots.