DNA Molecular Authentication And Protein Analysis Of Chinese Medicinal Materials And Herbal Slices Of Bombyx Batryticatus

Bombyx Batryticatus is dried fungus-infected complex,which is dead and stiffened by infection with Beauveria bassiana.Bombyx Batryticatus has functions of relieving convulsion,removing pain,phlegm and so on.It has muti-pharmacologic effects including phlegm,convulsion,convulsion and tetanus,etc.Adulterant products such as dead silkworm and dead silkworm infected with Metarhizium anisopliae were sold as Bombyx Batryticatus in the market.The quality of Bombyx Batryticatus has been affected a lot and it brings consumers some safe problems which cannot be ignored.This article established a convenient and effective method for identification of Bombyx Batryticatus.In addition,it revealed the changes of protein in processed Bombyx Batryticatus and highly-processed products by investigating the protein content,protein bands from SDS-PAGE and protein spots from 2-DE.This study can provide scientific basis and technical means for the quality control of Bombyx Batryticatus and its products,which can help to guarantee the safety and effectiveness of clinical use of the Bombyx Batryticatus.At the same time,it can open up a new way for the identification of special properties of animal medicines and lay a foundation for the study of the processing mechanism of Bombyx Batryticatus from the perspective of chemical change of biomolecules.The main parts of study are as follows:1.Firstly,mitochondrial genome sequences of three species,including Bombyx mori,Beauveria bassiana and Metarhizium anisopliae were used as targets.Suitableareas for designing species-specific primers were identified by DNAMAN software,and species-specific primers for identification of three species were then designed using Oligo 7.All samples were subject to genomic DNA extraction.Then PCR amplification wascarried out.The PCR amplification products were detected by agarose gel electrophoresis.All the primers were screened and the PCR conditions were optimized.The specificity and sensitivity of primers were investigated by using all the three specific primers.Finally,all the three sets of specific primers were used to detect the commercial samples.It was found that there was 1 counterfeit in processed Bombyx Batryticatus and 2 counterfeits in the highly-processed products.At the same time,the DNA template of the silkworm capsule contains Bombyx mori and Beauveria bassiana.It indicated that the main ingredient of the capsule is Beauveria bassiana Vuill.2.The extraction conditions of the protein were optimized.Optimize the methods of extracting proteins in Bombyx Batryticatus and using water as solvent,1:30 solid-liquid ratio,ultrasonic for 40 mins as the best extraction method.Using BCA method to determining the protein content of Bombyx Batryticatus included the processed products and highly-processed products by using optimal extraction method.It was found that protein content of the processed products was higher than that of highly-processed products.3.Using SDS-polyacrylamide gel electrophoresis?SDS-PAGE?to analyze protein of different batches in homemade or commercial processed and highly-processed Bombyx Batryticatus.And,the gel staining was performed by silver staining and coomassie brilliant blue staining.The results showed that there were 6bands in SDS-PAGE profile of homemade processed Bombyx Batryticatus and 2 of homemade highly-processed product.They could be considered as specific protein bands in processed and highly-processed Bombyx Batryticatus.There were 4 bands in SDS-PAGE profile of commercial processed Bombyx Batryticatus except P₄ and 2 of commercial highly-processed product.And,the results of SDS-PAGE staining by coomassie brilliant blue staining were consistent with that by silver staining.4.Using 2-DE to analysis the protein in different batches of processed and highly-processed Bombyx Batryticatus.The results showed that protein spots are mainly distributed at the molecular weight about 14.4 kD²5.0 kD and 30.0 kD⁶6.2 kD,pH in the range of 4⁷.The proteins were little distribution in alkaline area.There were 19 protein spots in processed Bombyx Batryticatus,which can be considered as the specific protein spots.There was a decrease in the number of protein spots after the processing of Bombyx Batryticatus.5.Proteins are digested with trypsin after being separated by SDS-PAGE.Peptides obtained are further analyzed by MALDI-TOF-MS.The corresponding peptide fragment fingerprinting is imported to the Mascot database to match with existing proteins.The results showed 2 reliable proteins.