High-level expression of a codon optimized recombinant dust mite allergen, Blo t 5, in Chinese hamster ovary cells
High-level expression of a codon optimized recombinant dust mite allergen, Blo t 5, in Chinese hamster ovary cells
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DOI：
10.1016/j.bbrc.2004.02.148
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发表时间：
2004-04-16
影响因子：
3.1
通讯作者：
Chua, KY
中科院分区：
文献类型：
--
作者：
Lim, LH;Li, HY;Chua, KY
Blo t5是室内尘螨Blomia tropicalis的主要过敏原。从完整的尘螨提取物中纯化天然Blo t 5（nBlo t 5）是冗长的并且产率低。在这项研究中，我们证明，密码子优化促进高水平表达的中国仓鼠卵巢（CHO）-K1细胞中的Blo t 5，从而允许生产足够的重组cBlo t 5的特异性免疫治疗。通过PCR合成密码子优化的Blot 5基因，并将其转染CHO K1细胞，经Geneticin（G418）稳定筛选。用过的培养基的Western免疫印迹分析检测到密码子优化的但不是野生型基因转染子的14 kDa阳性条带。此外，稳定的CHO-K1克隆产生高达13 mg/L的cBlo t 5蛋白，其具有与天然Blo t 5的人IgE反应性和变应原性的高度相关性。从而表明该重组形式的正确构象。(C)2004爱思唯尔公司All rights reserved.
Blo t 5 is a major allergen from house dust mite Blomia tropicalis. Purification of native Blo t 5 (nBlo t 5) from whole dust mite extract is tedious and gave low yield. In this study, we demonstrated that codon optimization facilitated high-level expression of Blo t 5 in Chinese hamster ovary (CHO)-K1 cells and thus allows production of sufficient recombinant cBlo t 5 for specific immunotherapy. A codon optimized Blo t 5 gene was synthesized by PCR and the codon optimized or wild-type Blo t 5 gene in pcDNA3.0 was transfected into CHO-K1 cells and stably selected with Geneticin (G418). Western-immuno blot analysis of spent culture media detected a positive band at 14 kDa for the codon optimized but not wild-type gene transfectants. In addition, a stable CHO-K1 clone produced up to 13 mg/L of the cBlo t 5 protein having a high correlation of human IgE reactivities and allergenicity to the native Blo t 5. thus indicating proper conformation of this recombinant form. (C) 2004 Elsevier Inc. All rights reserved.