白點症病毒對草蝦核醣體蛋白質表現之調節

Abstract

本研究利用兩階段超高速離心與不連續性蔗糖密度界面，建立由草蝦胃部純化核醣體 (ribosome) 之流程。經穿透式電子顯微鏡 (TEM) 檢視純化後的樣品，發現多核醣體的存在。以17.5 % 十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳 (SDS-PAGE) 分離組成核醣體之蛋白質，得到電泳分析圖譜。膠體經Coomassie Brilliant Blue R (CBR) 染色，將分子量介於10 kDa至 50 kDa之間的可見蛋白質帶切出，經蛋白質還原、烴基化反應與胰蛋白酶水解各個蛋白質帶，所得胜肽片段藉由液相層析串聯式質譜儀 (LC-nanoESI-MS/MS) 進行胺基酸定序。定序結果與 NCBInr資料庫比對後，於純化後樣品內鑑定出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以及RACK1等26個核醣體蛋白質。 此外，利用實驗室建構之草蝦EST資料庫，設計核醣體蛋白質S09、S11、S16、S24、L05、L08、L10、L10A、L23、L28、L32、L37之專一性核酸引子進行RT-PCR，分析白點症病毒 (white spot syndrome virus，WSSV) 感染前後，草蝦胃部核醣體蛋白質於基因層次上之改變。實驗發現草蝦胃部核醣體在WSSV感染前，蛋白質表現量最多，當WSSV感染2小時後，降至最低，隨後逐步攀升，感染後24小時至72小時表現量維持低水平。再以L23多株抗體分析感染WSSV後，核醣體蛋白質L23表現量之變化情形。L23於感染WSSV前表現量最多，12、24小時次之，感染48小時後L23含量驟降。証實核醣體蛋白質L23會因WSSV感染而改變其表現量。

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.