Massaer M, Mazzu P, Haumont M, Magi M, Daminet V, Bollen A, Jacquet A
Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, Gosselies, Belgium.
Int Arch Allergy Immunol. 2001 May;125(1):32-43. doi: 10.1159/000053794.
The major house dust mite allergen Der p 1 is associated with allergic diseases such as asthma. Production of recombinant Der p 1 was previously attempted, but with limited success. The present study describes the expression of recombinant (rec) ProDer p 1, a recombinant precursor form of Der p 1, in CHO cells.
As optimization of the codon usage may allow successful overexpression of protein in mammalian cells, a synthetic gene encoding ProDer p 1 was designed on the basis of the codon usage frequently found in highly expressed human genes. Gene synthesis was accomplished from a set of 14 mutually priming overlapping oligonucleotides and after two runs of polymerase chain reaction.
COS cells transiently transfected with the synthetic ProDer p 1 gene produced up to 5--10 times as much ProDer p 1 compared with the expression level obtained after transfection with the authentic gene. To stably express the recombinant allergen, CHO-K1 cells were transfected with the ProDer p 1 synthetic gene, and one amplified recombinant clone produced up to 30 mg of recProDer p 1 per liter in the culture medium before purification. recProDer p 1 was secreted as an enzymatically inactive single-chain molecule presenting three glycosylated immunoreactive forms of 41, 38 and 36 kD. When examined with respect to direct binding, recProDer p 1 and natural Der p 1 displayed very similar IgE reactivities. However, IgE inhibition and histamine release assays showed a much higher reactivity to natural Der p 1 compared to recProDer p 1.
These data indicated that codon optimization represents an attractive strategy for high-level production of allergen in mammalian cells.
主要的屋尘螨变应原Der p 1与哮喘等过敏性疾病相关。此前曾尝试生产重组Der p 1，但成效有限。本研究描述了重组（rec）ProDer p 1（Der p 1的一种重组前体形式）在CHO细胞中的表达。
由于密码子使用的优化可能有助于在哺乳动物细胞中成功过表达蛋白质，基于在高表达人类基因中常见的密码子使用情况设计了编码ProDer p 1的合成基因。基因合成由一组14个相互引发的重叠寡核苷酸完成，并经过两轮聚合酶链反应。
用合成的ProDer p 1基因瞬时转染的COS细胞产生的ProDer p 1比用天然基因转染后获得的表达水平高5至10倍。为稳定表达重组变应原，用ProDer p 1合成基因转染CHO - K细胞，一个扩增的重组克隆在纯化前在培养基中每升可产生高达30 mg的recProDer p 1。recProDer p 1作为一种无酶活性的单链分子分泌，呈现三种糖基化的免疫反应形式，分子量分别为41、38和36 kD。在直接结合检测中，recProDer p 1和天然Der p 1显示出非常相似的IgE反应性。然而，IgE抑制和组胺释放试验表明，与recProDer p 1相比，天然Der p 1的反应性要高得多。
这些数据表明密码子优化是在哺乳动物细胞中高水平生产变应原的一种有吸引力的策略。
