使用核醣體展示技術篩選對肺癌細胞特有的細胞表面單鏈抗 體和經由連接重鏈恆定序列改進單鏈抗體的親和力

Selection of single-chain variable fragments(scFvs) specific for A549 lung cancer cells using ribosome display and improvement of scFv affinity by linking constant heavy chain to the scFv

張政彥(Cheng-Yen Chang)

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(2015年)

摘要

本次實驗研究目的是要去建立單鏈抗體的核醣體展示庫，用於篩選與肺癌
細胞表面結合的單鏈抗體。單鏈抗體模板來源是從健康人人類周邊血分離出來
的單核球細胞mRNA 反轉錄所得到的cDNA 來作為模板，用PCR 的方式擴增
出抗體重鏈和抗體輕鏈並且組裝成VH/VL 單鏈抗體庫。這個單鏈抗體庫藉由小
麥胚芽蛋白質表達系統在體外轉錄、轉譯出蛋白質。為了分離出對A549 肺腺癌
細胞有特殊專一性的單鏈抗體，因此先對BEAS-2B 肺上皮細胞進行負向篩選，
再對A549 肺腺癌細胞做正向篩選。在五個回合篩選之後，用酶聯免疫吸附試驗
(ELISA)去挑選單鏈抗體，192 顆克隆中有四顆同時對於A549 和BEAS-2B 細
胞有較高的親和力，因此還需要進一步改良核醣體展示的實驗步驟，以篩選出
只對A549 細胞特有的細胞表面單鏈抗體。另一方面，因為單鏈抗體的應用有些
限制，如穩定性下降、半衰期短、對抗原的親和力不好，因此要改善這些問題
可以將單鏈抗體接上人類免疫球蛋白的重鏈恆定區域。在這個研究中我們成功
的將單鏈抗體接上人類免疫球蛋白的重鏈恆定區域序列，並且在大腸桿菌中表
達，將產物進行重新摺疊，最後測得scFv-Fc 的 Kd 值為70 pM，相較於scFv
親和力增加了178 倍。

關鍵字

核醣體展示技術 ； 單鏈抗體

並列摘要

The aim of this study is to construct a ribosome display library of single
chain variable fragments (scFvs) for screening lung cancer-binding scFvs.
mRNA was isolated from immunoglobulin transcripts derived by health people.
Heavy and light chain genes (VH and VL) were amplified separately by
RT-PCR and assembled into the VH/VL scFvs library. The scFv library was
transcribed and translated in vitro using a wheat germ protein expression
system. In order to isolate specific scFvs to A549 adenocarcinomic lung
cancer cells, negative selection on a lung normal epithelium cell line BEAS-2B
was carried out before positive selection on A549. After five rounds of
screening, cell enzyme-linked immunosorbent assay (ELISA) showed that four
of 192 scFv clones had high affinity to A549 cells. However, these scFvs also
displayed high affinity to BEAS-2B. In the current study, a comprehensive
human scFv library has been successfully constructed, however
further ribosome display selection is needed to screen for A549-specific scFvs.
On the other hand, because of some disadvantages of using scFvs, including
decreased stability, short half-life and decreased affinity to the antigen, one
approach in improving their function and affinity is to- the constant heavy chain
of human immunoglobulins to scFvs. In this study, we have successfully
joined a truncated human heavy chain constant fragment Fc region (the hinge
region, CH2 and CH3 domains) to an scFv, and the recombinant scFv-Fc
antibody was expressed in Escherichia coli and the resulting products were
refolded in vitro and characterized. The Kd of the scFv-Fc was estimated to be
70 pM, which is improved up to 178-fold in binding affinity compared to the
original scFv.

並列關鍵字

ribosome display ； scFv

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