Liang Ming-Hua, Zhou Shi-Shui, Jiang Jian-Guo
School of Biological Science & Engineering, South China University of Technology, Guangzhou, 510006, China; College of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China.
School of Biological Science & Engineering, South China University of Technology, Guangzhou, 510006, China.
Enzyme Microb Technol. 2017 Jul;102:74-81. doi: 10.1016/j.enzmictec.2017.04.001. Epub 2017 Apr 4.
HBscFv-IFNγ, a fusion protein constructed by fusing γ-interferon (IFNγ) with an antibody fragment HBscFv for the purpose of targeted delivery of the cytokine IFNγ, was designed in order to enhance its therapeutic efficacy through increasing its hepatoma localization. HBscFv and IFNγ were connected into HBscFv-IFNγ by the linker (GlySer), and then the multicopy recombinant plasmids pPICZαA/(HBscFv-IFNγ) were constructed and transformed into Komagatella (Pichia) pastoris X33. The engineering strain X4, which had much higher copy number and could secretively express HBscFv-IFNγ, was screened from transformed X33 by qPCR. Results from SDS-PAGE, Western blotting and ELISA indicated that HBscFv-IFNγ displayed an excellent immunoreaction against HBsAg. The culture supernatant of X4 was purified by 14F7 affinity chromatography to obtain the fusion protein HBscFv-IFNγ in a purity of 95-98%. The HBscFv-IFNγ was able to bind 27.9% HBsAg in the serum of HBV transgenic mice, showing that the antibody of HBscFv-IFNγ has high binding affinity against HBsAg. The expressing of the recombinant HBscFv-IFNγ in P. pastoris provides a promising and inexpensive diagnostic reagent for preventing HBV infection.
HBscFv-IFNγ是一种融合蛋白，它通过将γ干扰素（IFNγ）与抗体片段HBscFv融合构建而成，目的是实现细胞因子IFNγ的靶向递送，通过增加其在肝癌中的定位来提高其治疗效果。HBscFv和IFNγ通过连接子（GlySer）连接成HBscFv-IFNγ，然后构建多拷贝重组质粒pPICZαA/(HBscFv-IFNγ)并转化到巴斯德毕赤酵母X33中。通过qPCR从转化后的X33中筛选出拷贝数更高且能分泌表达HBscFv-IFNγ的工程菌株X4。SDS-PAGE、Western印迹和ELISA结果表明，HBscFv-IFNγ对HBsAg表现出优异的免疫反应。X4的培养上清液通过14F7亲和层析纯化，得到纯度为95%-98%的融合蛋白HBscFv-IFNγ。HBscFv-IFNγ能够结合HBV转基因小鼠血清中27.9%的HBsAg，表明HBscFv-IFNγ的抗体对HBsAg具有高结合亲和力。重组HBscFv-IFNγ在巴斯德毕赤酵母中的表达为预防HBV感染提供了一种有前景且廉价的诊断试剂。
