Please use this identifier to cite or link to this item:
標題: Generation and characterization of TG1(M13Δp3) and construction of plantibody expression system
作者: 林台珮
Lin, Tai-Pei
關鍵字: plantibody expression system
出版社: 分子生物學研究所
摘要: pep-3 is a peptide previously identified by phage display, which has a binding activity to a wide range of human cells and mouse myeloma cell Sp2/0-Ag-14 but not to serum proteins. In this study, I employed the cell binding property of pep-3 to establish a phage-display gene delivery system for mammalian cells. To achieve this goal, a DNA fragment containing the SV40 promoter, ecogpt coding region, and polyadenylation site (providing a selectable marker to monitor the transfection efficiency) was inserted into phagemids pCANTAB-B2 and pCANTAB-pep3. pCANTAB-B2 and pCANTAB-pep3 encode a fusion gp3 with a irrelevant peptide B2 and pep-3, respectively. The recombinant phages were obtained by transforming E. coli TG1 with the resultant plasmids pCANTAB-B2-gpt and pCANTAB-pep3-gpt separately, followed by superinfection with helper phage M13K07. The ability of pep-3 as the cell targeting for gene delivery was examined by incubating the recombinant phages with mouse myeloma cells Sp2/0-Ag14 followed by incubating the cells in the selective medium containing hypoxanthine, mycophenolic acid and xanthine. No survival cells were obtained in either of the recombinant phages used. One of the drawbacks of using phagemid/helper phage systems in phage display is the high background of phages not displaying the protein of interest. This may account for the failure in the gene transduction experiments. To overcome this problem, a defective M13K07 genome without g3 was constructed by PCR. Transformation of the construct into E. coli TG1 resulted in TG1(M13△p3). The feasibility of using TG1(M13△p3) as the host to produce recombinant phage was tested by examining the ability to release phage particles by Western blot analysis, ELISA, and spot test. The results indicated that TG1(M13△p3) was unable to produce phage particles, whereas phage particles were found in the supernatant of TG1(M13K07) transformed with phh3-Ars or pCANTAB-pep3-gpt, the plasmids encoding fusion gp3, although the titers were lower compared to that of TG1.
本實驗先前曾以噬菌體展示技術得到一個會與細胞結合,但對血清類可溶性蛋白不具結合力的多胜肽 (pep-3),本研究目的是利用pep-3與細胞結合之特性,嘗試建立一個以絲狀噬菌體為基礎的哺乳動物細胞基因傳送系統。為了篩選穩定的轉染細胞 (transfectant),首先把一段帶有 SV2-promoter-ecogpt 的 DNA 片段插入 pCANTAB-B2 及 pCANTAB-pep3 質體;前者表現之多胜肽與細胞無結合力,得到的質體分別稱為質體 pCANTAB-B2-gpt 與 pCANTAB-pep3-gpt。將此二質體分別送入 E. coli TG1 並以輔助噬菌體 M13KO7 進行感染,分別收集噬菌體顆粒,與哺乳細胞一起培養,之後加入含有 Hypoxanthine、Mycophenolic acid 與 Xanthine 的培養液,結果兩組沒有明顯的差異。由於以上述系統生產重組噬菌體無法得到純系的重組噬菌體,為了克服此問題,本研究首先把 M13KO7 基因組的 gp3 基因部分刪除,把此基因組送入 TG1,得到的細胞稱為 TG1(M13Δp3)。分別以 ELISA、西方點墨及點測試 (spot test) 確定此細胞無法表現 gp3 蛋白以及生產完整的噬菌體。為了證明此改造之 E. coli 可用於生產重組噬菌體,將表現外源蛋白與 gp3 融合蛋白的噬菌質體轉形至 TG1(M13Δp3) 後進行培養,收集此菌之培養液進行 spot test、西方點墨和 ELISA 分析,結果發現 TG1(M13Δp3) 能組裝出完整的噬菌體,但生產效率較低。此問題有待進一步去改善。
Appears in Collections:分子生物學研究所



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.