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目的克隆、杆状病毒/昆虫表达系统表达人源抗人干扰素α1b(huIFN-α1b)全抗体基因,获得全抗体蛋白。方法利用PCR技术以gIII-scFv融合表达的噬菌粒质粒为模板,分别克隆了5株抗体基因的轻链和重链;经Nco I/Xho I和Sac I/Hind III分别酶切,与经过相同酶切的pAC-K-CH3载体连接构建了昆虫细胞sf9表达载体pAC-K-CH3-H-L;经测序鉴定正确后,转染进入昆虫细胞sf9进行异源表达。采用免疫荧光(IFA)和ELISA对全抗体的表达和结合情况进行初步鉴定。采用Protein-A亲和层析柱对收获的全抗体IgG蛋白进行纯化,将纯化后的抗体蛋白进行SDS-PAGE电泳和Western blotting鉴定。结果成功扩增了人源抗人干扰素α1b抗体轻链和重链基因;免疫荧光(IFA)鉴定表明5株重组质粒在昆虫细胞sf9表达出了全抗体蛋白,ELISA鉴定表明其中3株与huIFN-α1b特异性结合;经Protein-A亲和层析柱纯化后每升细胞培养上清收获了5~20 mg的蛋白,Western blotting鉴定表明3株纯化的全抗体蛋白和huIFN-α1b特异性结合。结论通过杆状病毒/昆虫表达系统获得了3株人源抗人干扰素α1b(huIFN-α1b)全抗体蛋白。
Objective Cloning, baculovirus / insect expression system expression of human anti-human interferon α1b (huIFN-α1b) whole antibody gene to obtain full-antibody protein. Methods The phagemid plasmids expressing gIII-scFv fusion gene were used as templates to clone the light and heavy chains of five antibody genes respectively. The fragments were digested with Nco I / Xho I and Sac I / Hind III respectively, The same restriction enzyme pAC-K-CH3 vector was used to construct insect cell sf9 expression vector pAC-K-CH3-HL. After identification by sequencing, it was transfected into sf9 insect cells for heterologous expression. Immunofluorescence (IFA) and ELISA were used to identify the expression and binding of whole antibodies. The harvested antibody IgG was purified by Protein-A affinity chromatography. The purified antibody was identified by SDS-PAGE and Western blotting. Results The light chain and heavy chain genes of human anti-human interferon α1b antibody were successfully amplified. IFA showed that five recombinant plasmids expressed full-length antibody against sf9 in insect cells. ELISA showed that 3 of them were associated with huIFN -α1b. After purified by Protein-A affinity chromatography, 5 ~ 20 mg of protein was obtained per liter of cell culture supernatant. Western blotting showed that the purified proteins were specifically bound to huIFN-α1b . Conclusions Three human anti-human interferon alb (huIFN-α1b) full-antibody proteins were obtained by baculovirus / insect expression system.