利用核醣體展示技術結合次世代定序方法篩選及分析針對非小細胞肺癌表面具有親和力之胜肽

Selection and Analysis of Random Peptides Specific for Non-Small Cell Lung Cancer Cells (NSCLC) Using Ribosome Display and Next Generation Sequencing (NGS)

李宜宣(YI-HSUAN LI)

指導教授 ： 周正中

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2016年)

摘要

肺癌的發病率在癌症中排名第二，且在全球癌症死因中排名第一。而肺癌分為小細胞肺癌以及非小細胞肺癌。其中，非小細胞肺癌的患者佔肺癌的85%。由於目前並未研發出有效針對非小細胞癌細胞表面生物標記的標靶抗癌藥物，導致傳統的化學藥物治療效果並不顯著。因此，鑑定出能與非小細胞肺癌細胞表面具專一性結合的生物標記是發展精準癌症標靶治療的關鍵一步。為了找出非小細胞肺癌細胞表面專一性的生物標記，我們利用核糖體多胜肽展示技術，篩選出會與非小細胞肺癌表面結合的胜肽。隨機胜肽是由12個胺基酸所組成，藉由小麥胚芽蛋白質表達系統將隨機胜肽庫進行體外轉錄、轉譯後，先與人類正常肺細胞(BEAS-2B、MRC-5、HFL-1) 進行負向篩選，再與非小細胞肺癌細胞 (A549、H1299、H1650) 進行正向篩選。經由7次正負篩選後，再利用次世代定序 (next generation sequencing, NGS) 方法分析比較篩選的胜肽庫，目前已找出不與上述3個正常肺細胞株結合，但會和所有3個肺腺癌細胞株結合之胜肽羣。將此胜肽羣中挑選出現頻率最高之AH.1胜肽，利用cell-based ELISA以及免疫螢光染色法測試與細胞表面結合親和力之情形。

關鍵字

非小細胞肺癌 ； 核醣體展示技術 ； 胜肽

並列摘要

Lung cancer is the leading cause of cancer mortality worldwide. It falls into two categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter accounts for 85% of lung cancer. Current treatment for lung cancer such as chemotherapy obtains only limited success due to non-targeted drug delivery to the tumor site. Therefore, the discovery and identification of biomarkers on the surface of the cancer cells is a crucial approach toward tumor-specific precision therapy. In vitro ribosome display peptide library was used to identify specific peptides binding to the surface of NSCLC cells. The random peptide library (12 amino acids) was transcribed and translated in vitro using a wheat germ protein expression system. Negative selection on three normal human lung cell lines (BEAS-2B, MRC-5 and HFL-1) was carried out before positive selection on three NSCLC cell lines (A549, H1299 and H1650) to remove possible background noise. After seven rounds of negative-positive screening, we used next generation sequencing (NGS) to analyze those selected peptide libraries and have found a cluster of peptides specific to the binding of the above three NSCLS cell lines. We selected the most abundant peptide (named as AH.1) to perform cell ELISA experiments and immunofluorescence assay for validating the above finding.

並列關鍵字

NSCLC ； ribosome display ； peptide

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