Human IgG1 expression in silkworm larval hemolymph using BmNPV bacmids and its N-linked glycan structure
Human IgG1 expression in silkworm larval hemolymph using BmNPV bacmids and its N-linked glycan structure
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DOI：
10.1016/j.jbiotec.2008.09.013
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发表时间：
2009-01-01
影响因子：
4.1
通讯作者：
Ueda, Hiroshi
中科院分区：
文献类型：
--
作者：
Park, Enoch Y.;Ishikiriyama, Motoki;Ueda, Hiroshi
构建了表达人291J6 IgG重链和轻链的家蚕核多角病毒（BmNPV） bacmid，并将重组抗体分泌到家蚕幼虫血淋巴中。将重组半胱氨酸蛋白酶和几丁质酶缺失的BmNPV (BmNPV- cp—Chi(-)) bacmid/291J6 IgG注射到5龄家蚕幼虫的背部，约6天后采收。重组291J6 IgG的总产量为36 μ g/条幼虫，相当于8 mg/kg幼虫。重组抗体采用HiTrap rProtein a FF柱纯化至均匀性，纯化率为83.1%。经Western blot和酶联免疫吸附实验对纯化蛋白进行鉴定。用HPLC图谱法测定纯化蛋白的n链聚糖结构。家蚕幼虫产生并分泌的291J6 IgG糖蛋白的n -聚糖完全由Man α 1- 6man β 1- 4glcnac β 1-4(Fuc α 1-6)GlcNAc和Man α 1-6(Man α 1-3)Man β 1- 4glcnac β 1-4(Fuc α 1-6)GlcNAc两种低聚糖组成。(c) 2008 Elsevier B.V.版权所有
A Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expressing heavy and light chains of human 291J6 IgG was constructed and used to secrete recombinant antibody into silkworm larval hemolymph. Fifth instar silkworm larvae were reared and injected into the dorsum of the larvae with recombinant cysteine protease- and chitinase-deficient BmNPV (BmNPV-CP--Chi(-)) bacmid/291J6 IgG and harvested after approximately 6 days. The total yield of recombinant 291J6 IgG was 36 mu g/larvae, which is equivalent to 8 mg/kg of larvae. The recombinant antibody was purified to homogeneity using a HiTrap rProtein A FF column with a purification yield of 83.1%. The purified protein was identified by Western blot and ELISA experiments. The N-linked glycan structure of the purified protein was determined by the HPLC mapping method. The N-glycans of the 291J6 IgG glycoprotein produced in, and secreted by the silkworm larvae were composed exclusively of two kinds of paucimannose-type oligosaccharides, Man alpha 1-6Man beta 1-4GlcNAc beta 1 -4(Fuc alpha 1-6)GlcNAc and Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc. (c) 2008 Elsevier B.V. All rights reserved.