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。

關鍵字

澱粉磷解酶融合瘤技術；噬菌體表現系統

並列摘要

Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of the α-glucan in plant cells. In order to study the biological functions of starch phosphorylase, antibodies specific against SP have been produced by hybridoma technique. However, several problems inherited in this conventional technology were encountered during the generation of these anti-SP antibodies. Of which, the instability of anti-SP antibody-secreting hybrids was most frequently obsevered after several culture generations. Currently developed phage display antibody library technology represents an alternative way for the generation of biologically important antibodies. In 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 little somatic mutation. The reactivity and specificity of the recombinant anti-SP Fab fragments were confirmed by an enzyme-linked immunosorbent assay (ELISA), which is comparable to those of the parental anti-SP antibodies. These results suggest that 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.

並列關鍵字

Starch phosphorylase ； hybridoma technique ； phage display system

參考文獻

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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

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