Identification Of Ancient Silk Based On Proteomics And Immunological Techniques Proteomics

In recent years,the origin and spread of ancient silk which has attracted more and more attentions,was an unsolved mystery in archaeology.Silk can be divided into different species,including Bombyx mori,Antheraea Pernyi,Eri and Chestnut.Silks of different types exhibit different structures and properties.Analyses of different silk species and species identification of ancient silks are of great significance for studying the origin and spread of silks.In this thesis,modern silk samples were first analyzed by proteomics to obtain the biomarkers of different silk species.Then a method based on proteomics was established for the identification of silk species.By analyzing the amino acid sequences of different silks,the characteristic peptides were selected as the hapten,and coupled the carrier protein to obtain immunogenicity.Next,the specific antibodies were prepared through animal immunization to establish an antibody-based immunoassay for identifying silk species.Finally,the immunoassay was utilized to analyze the aging samples,which may provide a new perspective for the aging mechanism and theoretical basis for the ancient silk species identification.First,the silk fibroin in different silks was extracted with copper ethylenediamine solution,and the resultant protein was digested with enzyme in a solution.The protein after enzymatic hydrolysis was analyzed by LC-MS/MS to obtain the biomarkers for different species of silk: P05790 and P21828 for Bombyx mori;O76786 for Antheraea Pernyi;A0A0D5ZY13 for Eri;B6ZIV6 for chestnut.The established proteomic method was further applied to identify species of ancient silk,which can effectively find the biomarkers in the samples,confirming the effectiveness of the proposed method.Using the proteomic technique,the amino acid sequences of different species of silk were analyzed,and the characteristic peptides of Bombyx mori and Antheraea Pernyi silks were obtained.Different antibodies for Bombyx mori silk and Antheraea Pernyi silk were prepared(rabbit anti-Bombyx mori silk fibroin sequence antibody and rabbit anti-Antheraea Pernyi silk fibroin sequence antibody).Simultaneously,rabbit anti-Bombyx mori silk fibroin antibody and mouse anti-Bombyx mori silk fibroin antibody were prepared using silk fibroin powder as complete antigen.Bombyx mori silk was discerned from other species of silk by Western Blot.The antibody-based enzyme-linked immunosorbent assay(ELSIA)method was also used to identify silks,which can effectively separate Bombyx mori silk from Antheraea Pernyi silk.The species of ancient silks were successfully determined using immunoassays,which is consistent with the proteomics results.The Antheraea Pernyi silk fibroin powder was used as the complete antigen,and the rabbit anti-Antheraea Pernyi silk fibroin antibody was prepared through animal immunization.The artificially aged Antheraea Pernyi silk was obtained by heat aging and alkali aging,respectively,and the established immunological method was applied to detect them.Besides,various conventional analytical methods were employed to analyze the aging mechanism of Antheraea Pernyi silk.It was found that the aging degree induced by alkali was higher than that by heat aging.In addition,ELISA was more sensitive than immunoblotting in analyzing aged samples.To sum up,proteomics and immunological methods were established for identifying ancient silks.The new methods may provide new analytical tools for the identification of ancient silks and new perspective on exploring the origin and dissemination of silks.