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Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of t

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對澱粉磷解脢具有特異性的老鼠單株重組抗體片段之產生與 其特性分析

澱粉磷解酶 (starch phosphorylase ； SP) 是植物體內參與澱粉代謝的 重要酵素之一。其主要的功能為催化 α-1,4 葡萄聚醣 (α-1,4 glucan) 可 逆性的磷解反應，亦即從葡萄聚醣之非還原端移出或加入一個葡萄醣 單位。為了研究澱粉磷解酶的功能，先前已有人利用融合瘤技術來製 造出對澱粉磷解酶具有特異性的抗體。但是由此技術來製造出的抗體 產生了一些問題，如融合瘤細胞經過數代培養之後，細胞並不穩定，

無法再分泌抗體甚至造成死亡。為了克服上述的問題，噬菌體表現系 統提供了一種方法來產生大量對 SP 抗原具有特異性的單株抗體。在 本研究中，我們利用 PCR 的方式來大量增殖重鏈和輕鏈的產物，並 將其接到質體上，並表現在的 M13 噬菌體的表面上。進一步由西方 墨點分析法可偵測到 50 kDa 大小的重組蛋白質。 DNA 序列分析結果 顯示重鏈和輕鏈都有被表現出來，並與基因庫比對發現有少部分的突 變位置。而藉由 ELISA 觀察 anti-SP Fab 也具有與 SP 結合之特異性。

由此可顯示說噬菌體表現系統提供了另一種方法來製造出特異性的抗 體，並也解決了利用融合瘤細胞來製造特異性抗體所產生的問題。

(2)

Production and Characterization of Mouse Monoclonal Fab Antibodies Specific to Starch Phosphorylase

Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of t

he α-glucan in plant cells. In order to study the biological functions of starch phosp

horylase, antibodies specific against SP have been produced by hybridoma techniqu

e. However, several problems inherited in this conventional technology were encou

ntered during the generation of these anti-SP antibodies. Of which, the instability of

anti-SP antibody-secreting hybrids was most frequently obsevered after several cult

ure generations. Currently developed phage display antibody library technology rep

resents an alternative way for the generation of biologically important antibodies. I

n this study, we amplified both heavy and light genes from anti-SP hybridoma cells

, which were later cloned and expressed on the surface of M13 phage. Recombinant

Fab recognizing starch phosphorylase was produced and detected as a 50 KDa band

on the western blots. Nucleotide sequence analysis indicated that the cloned heavy

and light genes were derived from certain immunoglobulin germline genes with littl

e somatic mutation. The reactivity and specificity of the recombinant anti-SP Fab fr

agments were confirmed by an enzyme-linked immunosorbent assay (ELISA), whi

ch is comparable to those of the parental anti-SP antibodies. These results suggest t

hat the phage display antibody system may provide a better way for the generation

of specific antibodies and for the rescue of genetically unstable antibody-secreting

hybridoma cells.

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