Detection of Tea Astringency and Generation of Antibody against Two Active Ingredients in Danshen via Artificial Oil Bodies
| Autor: | Yu-En Shih, 施毓恩 |
|---|---|
| Rok vydání: | 2019 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 107 Oil body is an organelle in plant seeds which store neutral lipids as energy source for seed germination and subsequent growth. In natural, it constitutes with surface oil body proteins, i.e., oleosin, caleosin and steroleosin, monolayer phospholipids (PLs) and interior triacylglycerols (TAGs) matrix. These proteins provide electronegative repulsion and steric hindrance that keep seed oil body stabilize. In previous researches, the artificial oil bodies (AOBs) can be constituted by sonication oleosin/caleosin, PLs and TAGs, and develop several biotech applications, e.g., drug delivery system and enzyme immobilization. In this dissertation, I separately use gene engineering and chemical modification on caleosin for new biotech development. Caleosin has been proposed 2 hydrophilic domains and 2 hydrophobic domains, α-helix and proline knot. In previous research show the recombinant caleosin without hydrophobic α-helix domain has better over-expression quantity in E. coli. and the AOB constituted with this protein shows similar stability with natural form. In first chapter, one saliva protein, Histatin 3 (Hst 3), which bind with tea polyphenol and cause the sensory of astringent was genetically engineered with recombinant caleosin. The AOB constituted with caleosin-Hst 3 fusion protein showed the tea polyphenols caused these AOB aggregation and form oily floatation layer on the aqueous surface. The thickness of AOB aggregated floatation layer showed the positive correlation with epigallocatechin gallate (EGCG) contents in tea infusion and the score of sensory evaluation. It appears this engineered caleosin-Hst3 formed AOB can be a new method for in vitro astringent assay. In the second chapter, the therapeutic compounds lithospermic acid B (LSB) and rosmarinic acid (RA) in Salvia miltiorrhiza (Danshen), were conjugated to recombinant caleosin via amide bonding. The modified caleosin formed stable AOB as previous researches and presented small molecule hapten on the surface. Polyclonal antibodies against LSB and RA were successfully produced in hens injected with AOB constituted with each of the LSB and RA modified caleosin. These polyclonal antibodies showed small compounds specificity and was further developed into indirect competitive ELISA (icELISA). It appears this AOB hapten presenting system is a suitable method for the production of antibodies against small molecules. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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