硫氧还蛋白抗体柱的制备

目的:探索硫氧还蛋白(Trx)抗体柱对Trx融合蛋白纯化的可行性。方法与结果:对含有Trx基因的质粒表达载体pTrxFus进行改造,在Trx读框之后加入6×His序列,并在大肠杆菌中表达C端带有6×His标签的Trx,经Ni2+柱亲和纯化后制备多克隆抗体;把经蛋白A纯化后的抗体偶联在溴化氰活化的琼脂糖凝胶上,制成Trx抗体柱;用此抗体柱纯化与Trx融合表达的豇豆胰蛋白酶抑制剂(CpTI),SDS-PAGE结果显示获得了纯度较高的Trx-CpTI。结论:用Trx抗体制成的免疫亲和层析柱可以有效纯化Trx融合蛋白。

Preparation of Anti-Thiroredoxin Antibody Column

Objective：To develop an approach for the purification of thioredoxin（Trx）-fused recombinant proteins.Methods ＆ Results：We expressed recombinant Trx with 6×His tag at its C terminal in the E.coli GI174 by inserting 6×His codes into the downstream region of the Trx ORF on plasmid pTrxFus.The recombinant Trx was purified by affinity with the Ni2＋-agarose and then applied to immunize the rabbits to produce polyclonal antibody.The antiserum with a titer of 1∶100 000 assayed by indirect ELISA was subjected to protein A sepharose for the purification of IgG.The purified IgG was immobilized to sepharose 4B by the cyanogen bromide method to prepare an anti-Trx antibody affinity column.A Trx-fused protein Trx-CpTI（cowpea trypsin inhibitor fused to Trx） was effectively purified by the antibody column.Conclusion：The immunoaffinity chromatography with anti-Trx antibody column provided an effective way for the purification of recombinant Trx-fused proteins