抗重组GST单克隆抗体的制备及其在融合蛋白纯化中的应用

Preparation of monoclonal antibody against recombinant GST and its application in purification of GST fusion protein

目的制备抗重组GST的单克隆抗体(mAb), 并用来纯化重组GST融合蛋白。方法用含重组GST融合蛋白基因的pGEX4T -1质粒转化E.coli BL21，IPTG诱导GST融合蛋白表达，亲和层析和凝胶过滤法分离表达的重组GST融合蛋白。以此蛋白作为抗原，免疫Balb/c小鼠，按传统的杂交瘤技术制备mAb。将抗GST mAb经Protein A纯化后，与Sepharose 4B偶联。结果经3次亚克隆后，获得两株分泌抗GST载体特异性mAb的杂交瘤。采用该mAb对两种不同的GST融合蛋白进行亲和层析纯化后，经SDS-PAGE鉴定达到了商品化Glutathione-Resin的亲和层析纯化效果。结论用抗GST蛋白特异性mAb亲和层析纯化融合蛋白是一种经济、实用的方法，且可用于Glutathione-Resin亲和层析纯化后的二次纯化。

Aim To prepare and characterize a monoclonal antibody against recombinant glutathione S-transferase(GST) for purifying GST fusion protein. Methods The GST-follistatin fusion protein was expressed by using a pGEX4T-1 expression vector in Escherichia coli BL21 and purified by glutathione-resin affinity column chromatography. Then female Balb/c mice were immunized with the GST-FS, The immunized splenocytes were fused with NS-1 hybridoma cells. Dreparation of the mAb was used by conventional hybridoma techniqal. The mAb purified by protein A, was culpled with Sepharose4B to purify further GST fusion protein by affinity chromatography. Results The SDS-PAGE showed that the GST fusion protein could be purified effctively by specific mAb affinity chromatography as same as by glutathione-resin affinity chromatography. Conclusion mAb affinity chromatography will be a ecnomical and useful method and it can be used for secondary purification of GST fusion protein following glutathione-resin affinity chromatography.