臺灣博碩士論文加值系統

治療性的蛋白質藥物在製造上需要被大量生產且最終折疊成具有生物活性的形式。蛋白質合成過程的關鍵因子包括mRNA 5’端的結構，核糖體密度與tRNA豐富度；而在其中tRNA豐富度仍不是完全清楚。在轉譯期間，同義密碼對應到tRNA同功受體的機率並不同，可轉譯出相同胺基酸，此現象稱為密碼子使用偏好，而這種使用偏好在不同物種間有顯著的差異。過去文獻已經顯示tRNA豐富度與密碼使用率或tRNA基因重複數有關連性；然而，這些研究結果並無一致性而且取決於物種個體。因此，我們想要探討在中華倉鼠細胞株內，tRNA基因重複數，密碼使用率與tRNA豐富度在蛋白質表現效率上的影響。首先，我們建立三個不同介白素-2基因，即具有不同mRNA序列，皆可轉譯出相同胺基酸序列。第一個根據tRNA基因重複數少與密碼使用率高的密碼做改變，另一個以tRNA基因重複數多與密碼使用率低的做修改，最後一個根據tRNA基因重複數多且密碼使用率高的密碼做修飾。我們以酵素連結免疫法分析於培養液中被分泌的介白素-2，比較改變tRNA基因重複數與密碼使用率後的蛋白質表現量。將介白素-2修飾為tRNA基因重複數少與密碼使用率高者表現量較更改為tRNA基因重複數多與密碼使用率高者低，但是其表現量又比改變為tRNA基因重複數多與密碼使用率低者高。除了介白素-2更改為tRNA基因複製數多且密碼使用率高者外，其他組之介白素相比於野生型皆無明顯改變。利用外送丙胺酸、纈草胺酸、羥丁胺酸tRNA的方式提高tRNA的表現量後，發現介白素-2的蛋白表現量沒有顯著差異。接下來，我們以次世代定序系統測定中華倉鼠細胞株的tRNA，透過CLC Genomics Workbench軟體分析，結果顯示細胞內所含的tRNA近似於密碼使用率。綜合以上的結果，tRNA密碼使用，而非基因重複數，可能直接影響到tRNA豐富度於哺乳類動物細胞中。

Therapeutic protein drugs need to be produced in large amount in their biological active form. Key factors in protein synthesis include the 5’ end structure of mRNA, ribosomal density and tRNA abundance; in addition, tRNA abundance remains poorly understood. During translation, synonymous codons adapted to tRNA isoacceptors are translated into the same amino acid with different frequencies, a phenomenon termed codon-usage bias which is dramatically different among species. Previous reports have revealed the possible correlation between tRNA abundance and tRNA gene copy number or codon usage; however, the results are inconsistent and depended on the organisms. Therefore, we want to study the effects of tRNA gene copy number, codon usage and tRNA abundance on protein expression efficiency in Chinese hamster ovary (CHO) cells. First, we created three interleukin-2 (IL-2) genes containing the same amino acid sequence but with different codons (different mRNA sequences). One is modified according to low tRNA gene copy number and high codon usage, another is changed based on high tRNA gene copy number and low codon usage, and the other is altered to codon with high tRNA gene copy number and high codon usage. To compare expression level of codon usage- and gene copy number-changed IL-2, we determined the amount of secreted IL-2 in medium with enzyme-linked immunosorbent assay. IL-2 with low tRNA gene copy number and high codon usage was expressed at lower level than IL-2 with altered high tRNA gene copy number and codon usage, but it expressed higher level of IL-2 than that with changed to high tRNA gene copy number and low codon usage. Except for the group of IL-2 with high tRNA gene copy number and high codon usage, no dramatic changes of IL-2 expression in other groups were observed compared to wild type. Enhancing tRNA expression, by transfection of tRNA for valine, alanine, and threonine, did not lead to significant change in IL-2 protein expression. Next, we investigated tRNA content in CHO cells by next generation sequencing and used CLC Genomics Workbench software for analysis. The results demonstrated that tRNA abundance is depended on codon usage as a proxy in CHO cells. Taken together, selection of codon usage, rather than tRNA gene copy number, may directly affect tRNA abundance in mammalian cells.

目錄
緒論
一、蛋白質藥物的發展 1
二、影響蛋白質合成的因素 4
三、tRNA豐富度的探討 6
四、研究目標與策略 9

材料與方法
一、細胞培養 (Cell culture) 10
二、質體製備 (Plasmid preparation) 15
三、質體構築 (Plasmid construction) 19
四、染色體DNA萃取 (Genomic DNA extraction) 24
五、反轉錄聚合酶鏈鎖反應 (RT-PCR) 26
六、聚合酶鏈鎖反應 (PCR) 29
七、西方點墨法 (Western blot) 30
八、酵素連結免疫吸附法 (Enzyme-linked immunosorbent assay; ELISA) 38
九、次世代定序分析 (Next generation sequencing analysis) 40

實驗結果
一、根據密碼使用率與tRNA基因重複數介白素-2的改變密碼是否影響其蛋白質表現量 42
二、過量表現特定tRNA是否會影響介白素-2的蛋白質表現量 43
三、tRNA豐富度實際在中國倉鼠細胞株的分佈 43

討論 45
結論 49
參考文獻 50
附表 62
附圖 72
附註
附註一、中國倉鼠細胞株的密碼使用率 (codon usage) 83
附註二、中國倉鼠細胞株於染色體上的tRN A重複數 84

表目錄
表一、質體建築PCR反應引子 62
表二、反轉錄聚合酶鏈鎖 (RT-PCR)反應引子 63
表三、即時定量聚合酶鏈鎖反應 (real-time PCR)反應引子 64
表四、介白素-2 (LH) 所更改的密碼與對應反密碼 65
表五、介白素-2 (HL) 所更改的密碼與對應反密碼 66
表六、介白素-2 (HH)所更改的密碼與對應反密碼 67
表七、三組介白素-2實際更改比例 68
表八、三組介白素-2密碼更改後的序列 69
表九、次世代定序分析結果與資料庫的對照表 70

圖目錄
Figure 1. 建築分泌型的EGFP於pIRES載體 72
Figure 2. 根據單獨密碼使用率高或tRNA基因重複數改變介白素-2的密碼並不會影響其蛋白質表現量 74
Figure 3. 過度表現tRNA並不會影響介白素-2的蛋白質表現量 77
Figure 4. 中國倉鼠細胞的tRNA豐富度相近於密碼使用率 80

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