碩士 / 國立臺灣大學 / 動物學研究研究所 / 94 / In this study, we established a high throughput and prompt procedure to purify the native form of ribosomes from Penaeus monodon stomach tissues by means of two-steps ultracentrifugation and discontinuous sucrose density phase. Using Transmission Electron Microscope (TEM) to observe purified protein samples, we discovered the polysome structure in it. These ribosomal proteins were separated by 17.5 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile. The visible bands of protein profile between 10 ~ 50 kDa were excised from the Coomassie Brilliant Blue R stained gel, and then following trypsin digestion to reduce and alkylate ribosomal proteins in those bands. Those peptide fragments were ran through liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nano-ESI-MS/MS) using quadrupole/time-of-flight mass spectrometers to gather the peptide sequence information. We compared those peptide sequence data with the nonredundant database in the National Center for Biotechnology Information, and identified 26 ribosomal proteins : S1, S3, S3a, S4, S5, S9, S10, S11, S13, S14, S16, S18, S20, S23, S25, S27a, S28, P0, L11, L12, L23a, L24, L30, L34, L38, and the Receptor for activated C-Kinase (RACK1).
In order to analysis the alternation of ribosomal protein expression in WSSV infected Penaeus monodon, We gathered the cDNA sequences from EST database to design specific primers for ribosomal proteins S09, S11, S16, S24, L05, L08, L10, L10a, L23, L28, L32, and L37. After
WSSV infection, the RT-PCR results showed that ribosomal protein expression dropped to a bottom line at 2 hpi (hours post infection), then the expression quantity climbed up until 24 hpi. Between 24 hpi to 72 hpi, the ribosomal proteins expression quantity kept at a lower level then 0 hpi. Following the RT-PCR, using polyclonal antibody against L23 ribosomal protein to detect the alternation expression quantity after WSSV infection in the purified ribosomal sample, We discovered that L23 expression were downregulated at 12 and 24 hpi, even vanished at 48 hpi. We demonstrated that ribosomal protein L23 expression were altered after WSSV infection.
| Identifer | oai:union.ndltd.org:TW/094NTU05312007 |
| Date | January 2006 |
| Creators | Tai-Jung Wu, 吳岱融 |
| Contributors | Guang-Hsiung Kou, Chu-Fang Lo, 郭光雄, 羅竹芳 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 67 |
Page generated in 0.024 seconds