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Construction of HBV expression plasmids with mutation in putative hnRNP A1 site(nt243-248)

Construction of HBV expression plasmids with mutation in putative hnRNP A1 site(nt243-248). 實驗室：榮總教研部分生實驗室 指導教授：蘇宗笙. 目的.

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Construction of HBV expression plasmids with mutation in putative hnRNP A1 site(nt243-248)

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  1. Construction of HBV expression plasmids with mutation in putative hnRNP A1 site(nt243-248) 實驗室：榮總教研部分生實驗室 指導教授：蘇宗笙

  2. 目的 • HBV是個很小的病毒，但是卻可以製造很多種蛋白質，由學姐的論文已找出可能為HBV的ESE(exonic splicing enhancer)nt470-478和ESS(exonic splicing silencer)nt231-236，而最近的研究報告指出可能為hnRNP A1 site應在nt231-236稍後，和HIV比對後，估計應在nt243-248，故實驗目的在於驗證nt243-248是否為ESE或ESS。

  3. 方法 • 改變nt243-248“tagaga“為互補序列“atctct“ • QuickChange Site-Directed Mutagenesis • 將做好的pSVHBV放入癌細胞後抽取生成的RNA

  4. 實驗過程 • 製作大量plasmid以備用。 pSVHBV pHBV(-9/1004) pHBV(-9/1004)∆ESE pHBV(-9/1004)∆ESS • 依據QuickChange Site-Directed Mutagenesis步驟，用PCR製作plasmid，sequencing。 pHBV(-9/1004)c(243-248) pHBV(-9/1004)∆ESEc(243-248) pHBV(-9/1004)∆ESSc(243-248)

  5. ☼發現學姐先前製作的pHBV(-9/1004)∆ESS缺少了一base pair，故仍依QuickChange Site-Directed Mutagenesis製作新的pHBV(-9/1004)∆ESS。 • 以pSVHBV為vector，用enzyme SalI-9-PshAI498製作plasmid，sequencing。 pSVHBVc(243-248) pSVHBV∆ESEc(243-248) pSVHBV∆ESSc(243-248) ☼原本enzyme用BsaBI528，但切不動，故改用PshAI498。 • 暑假結束，transfection之後的實驗由學長接著進行。

  6. 心得 • 覺得榮總實驗室的氣氛很融洽，學長姐也很熱心的指導我做實驗。 • 做實驗前實驗步驟要先訂好，實驗的藥品要先計算好，以免造成不必要的浪費。 • 實驗設計需要對照組，且應考慮各個可能的狀況設計不同的對照組，實驗有問題時才能解決。 • 實驗因常需要等待，故在做實驗前應先做好時間的分配，以免要半夜做實驗。

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