建立可溶性單鏈抗體表現與純化系統

Abstract

能專一性鑑別新型H7N9流感病毒血球凝集素之單株抗體1C6B的單鏈抗體，於大腸桿菌中表現時，幾乎都形成了不溶的包涵體。相反的，表現1C6B-scFv與麥芽糖結合蛋白之融合基因時，則可形成可溶性之融合蛋白，但它卻喪失了與H7N9 HA結合的能力。因此，本研究旨在生產可溶形式的1C6B-scFv，並且使其仍具有正常之功能和專一性，並藉此建立生產可溶性scFv之蛋白質表現與純化系統。實驗結果顯示，從MBP融合蛋白切割出之1C6B-scFv可溶性佳，並且可以利用Ni-NTA及DEAE層析管柱進行純化。更重要的是，可溶形式的1C6B-scFv保留了與H7N9 HA結合的能力。本研究亦利用此步驟表現及純化出可溶形式之anti-NP-scFv與anti-NS1-scFv，並且使其仍保有辨認NS1與NP之專一性結合能力。因此，本研究利用MBP表現系統生產MBP-scFv，並進一步以TEV protease剪切及管柱層析法來純化可溶形式之scFv，成功建立了可快速獲得可溶性且具有與其原始全長抗體相同專一性之單鏈抗體的表現及純化系統。

The single-chain variable fragment (scFv) of the monoclonal antibody 1C6B, which is specific for recognition of the novel H7N9 influenza virus hemagglutinin (HA), was mainly expressed as inclusion bodies in Escherichia coli. In contrast, the maltose binding protein (MBP)-fused 1C6B-scFv was expressed as a soluble form, but it lost the capability for binding with H7N9 HA. Thus, the present study aims to produce the soluble form of 1C6B-scFv for characterization of the function and specificity of 1C6B-scFv, and establish a general protein expression and purification system which can be applied in generating soluble scFvs. The results showed that the 1C6B-scFv cleaved from the MBP fusion protein becomes soluble and can be easily purified by Ni-NTA and DEAE agarose chromatography columns. More importantly, the soluble form of 1C6B-scFv retains its capability for binding with H7N9 HA. By using the same procedure, the soluble form of anti-NP-scFv and anti-NS1-scFv were also purified, and they retained the capability for binding with NP and NS1, respectively. This study successfully established a protocol, composed of the MBP-scFv expression system, the TEV protease cleavage step, and the column chromatography, to produce the soluble scFv, which performs the same antigen binding specificity as the original full-length antibody does.