HBscFv–IFNγ在毕赤酵母X33中的表达、纯化及鉴定

Expression Purification and Verification of HBscFv-IFNγ in Pichia pastoris x33

摘要:

为了有效治疗乙肝病而研究了将抗乙肝病毒表面抗原单链抗体(single-chain Fv against HBV surface antigen, HBscFv)与临床治疗乙肝常用的γ-干扰素(γ-interferon, IFNγ)连接的融合蛋白(HBscFv-IFNγ)。采用重叠PCR法将基因hbscfv与ifnγ连接成hbscfv-ifnγ, 再构建成多拷贝重组质粒pPICZαA/(hbscfv-ifnγ)1,2,4, 然后转入巴斯德毕赤酵母X33。从中筛选出的工程菌株X4能够分泌表达目的蛋白HBscFv-IFNγ, 并用SDS-PAGE、Western blotting和ELISA方法进行了初步鉴定, 结果表明组成HBscFv-IFNγ的HBscFv和IFNγ仍具有生物学活性。用14F7亲合层析纯化X4的发酵液可获得纯度达95%~98%的HBscFv-IFNγ。它可中和HBV转基因小鼠血清中27.9%的乙肝病毒表面抗原(HBV surface antigen, HbsAg), 这表明HBscFv-IFNγ上的抗体能够与生物体内的HBV有效结合。可见, HBscFv-IFNγ将是一种防治乙肝病而有开发前景的靶向新药。

Abstract:

In order to effectively cure hepatitis B virus (HBV), we studied on fusion protein HBscFv-IFNγ, which was connected with single-chain Fv against HBV surface antigen(HBscFv) and γ-interferon(IFNγ) of being used in clinic against HBV. Adopting overlap PCR, the hbscfv and the ifnγ were connected into hbscfv-ifnγ. Then the pPICZαA/(hbscfv-ifnγ)1,2,4 of multi-copy recombinant plasmid were constructed and transformed into Pichia pastoris x33. The engineering strain x4 was screened from transformed x33 and could secretively express HBscFv-IFNγ. The preliminary verification indicates that HBscFv-IFNγ has the bioactivity of HBscFv and IFNγ by SDS-PAGE, Western blotting and ELISA. The supernatant of culturing X4 was purified by 14F7 affinity chromatography to HBscFv-IFNγ with purity of 95%~98%. The HBscFv–IFNγ is able to bind 27.9% HBV surface antigen (HBsAg) in the serum of HBV transgenic mice, which shows the antibody of HBscFv-IFNγ has bioactivity in vivo. Therefore HBscFv-IFNγ can shed light on the development of a new promising HBV -targeted drug.