Lim L H, Li H Y, Cheong N, Lee B W, Chua K Y
Department of Paediatrics, National University of Singapore, Singapore.
Biochem Biophys Res Commun. 2004 Apr 16;316(4):991-6. doi: 10.1016/j.bbrc.2004.02.148.
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-immunoblot analysis of spent culture media detected a positive band at 14kDa 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.
Blo t 5是热带屋尘螨的一种主要变应原。从全尘螨提取物中纯化天然Blo t 5（nBlo t 5）既繁琐又产量低。在本研究中，我们证明密码子优化有助于Blo t 5在中国仓鼠卵巢（CHO）-K1细胞中高水平表达，从而能够生产足够的重组cBlo t 5用于特异性免疫治疗。通过PCR合成了密码子优化的Blo t 5基因，并将pcDNA3.0中的密码子优化型或野生型Blo t 5基因转染到CHO-K1细胞中，并用遗传霉素（G418）进行稳定筛选。对用过的培养基进行的蛋白质免疫印迹分析在14kDa处检测到一条阳性条带，该条带对应于密码子优化型基因转染细胞，而非野生型基因转染细胞。此外，一个稳定的CHO-K1克隆产生的cBlo t 5蛋白高达13mg/L，其与人IgE反应性和对天然Blo t 5的变应原性高度相关，这表明该重组形式具有正确的构象。
