本文關(guān)鍵詞：人CD59活性位點(diǎn)短肽封條的抗腫瘤效應(yīng)的研究　出處：《青島大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 噬菌體展示 全細(xì)胞篩選 CD59蛋白 腫瘤逃逸 短肽封條 活性位點(diǎn)篩選

【摘要】： 目的本研究旨在通過噬菌體展示技術(shù)尋找與腫瘤逃逸相關(guān)的CD59活性位點(diǎn)，研制特異性針對(duì)CD59活性位點(diǎn)的短肽封條，觀測(cè)其封閉或干擾效應(yīng)，為腫瘤的靶向免疫治療開辟一條新途徑。 方法采用噬菌體隨機(jī)十二肽庫(kù)展示技術(shù)對(duì)Hela細(xì)胞裂解蛋白及層析純化后的CHO細(xì)胞的裂解蛋白進(jìn)行5輪親和淘洗篩選；從篩選后的噬菌體庫(kù)中分別隨機(jī)挑選15／16個(gè)噬菌體克隆進(jìn)行測(cè)序，并對(duì)隨機(jī)挑選的噬菌體克隆與野生型M13噬菌體進(jìn)行競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)；經(jīng)序列分析找出共享序列并推斷其相應(yīng)的氨基酸位點(diǎn)，，同時(shí)與CD59活性區(qū)域進(jìn)行分析比對(duì)，找出人CD59分子的活性位點(diǎn)，并合成短肽封條，觀測(cè)其封閉效應(yīng)。 結(jié)果經(jīng)過5輪全細(xì)胞篩選后，兩種噬菌體庫(kù)的回收量均可達(dá)到10~7～10~8數(shù)量級(jí)，且競(jìng)爭(zhēng)結(jié)合分析及細(xì)胞特異性結(jié)合分析顯示篩選后的噬菌體克隆及噬菌體庫(kù)對(duì)兩種細(xì)胞蛋白均具有明顯的特異性結(jié)合效應(yīng)。通過測(cè)序結(jié)果推斷出Hela細(xì)胞表面CD59的多肽序列(—ACHWPWCHGC—)與CHO細(xì)胞表面CD59的多肽序列(—ACHWWHAWTC—，—ACWWFHPHLC—)，并利用高同源性的Hela細(xì)胞的CD59短肽序列為模型合成高特異性的短肽封條。 結(jié)論利用噬菌體肽庫(kù)對(duì)兩種細(xì)胞進(jìn)行全細(xì)胞蛋白篩選得到了與CD59特異性結(jié)合的短肽序列，該短肽序列可能是位于腫瘤細(xì)胞表面CD59的配體蛋白。經(jīng)活性檢測(cè)證實(shí)該短肽封條對(duì)人CD59有明顯的封閉作用。
[Abstract]:Objective The aim of this study was to find CD59 active site related to tumor escape through phage display technique , and to develop a short peptide seal specific for CD59 active site , and to observe its blocking or interference effects , which opened a new way for targeted immunotherapy of tumors . Methods A phage random twelve peptide library was used to screen the lysate of Hela cell lysate and purified CHO cells . 15 / 16 phage clones were randomly selected from the screened phage library for sequencing , and the random selection phage clones were randomly selected to compete with the wild type 13 phage . The sequence analysis was used to identify the corresponding amino acid sites and to identify the active sites of CD59 molecules and to synthesize short peptide seals to observe the blocking effect . Results After five rounds of whole cell screening , the recovery of two phage libraries could be up to 10 ~ 7 ~ 10 ~ 8 orders of magnitude , and the competitive binding analysis and cell - specific binding assay showed that the phage clones and phage libraries had obvious specific binding effect on both cell proteins . The polypeptide sequences of CD59 ( ACHWPW CHGC - ) and CD59 on the surface of CHO cells were deduced by sequencing . The high - specific short peptide seal was synthesized by using the CD59 short peptide sequence of Hela cells with high homology . Conclusion The short peptide sequence which specifically binds CD59 is obtained by using phage peptide library to screen two kinds of cells . The short peptide sequence may be a ligand protein located on the surface of tumor cell CD59 . The activity test proves that the short peptide seal has obvious blocking effect on human CD59 .

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392;R730.5

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本文編號(hào)：1400488