基于巴斯德毕赤酵母表达系统探讨了屋尘螨过敏原Der p 1的重组表达与纯化.首先,采用毕赤酵母GS115表达密码子优化的全长编码基因PreProDer p 1,其产量可达100 mg/L,进一步共表达分子伴侣实现产量提高到 140 mg/L,并实现 3L反应器发酵产量提高到 1 g/L.其次,对上述重组蛋白发酵液分别使用阳离子交换层析及亲和层析进行了纯化工艺优化,目的蛋白得率达到60.7%.本研究为后续PreProDre p 1诊断试剂盆的开发提供了参考.
Recombinant Expression and Purification of House Dust Mite Allergen Der p 1 in Pichia pastoris
Dust mite allergen is one of the most common allergens that cause allergies in humans.Der p 1 protein,the first allergen of house dust mite(Dermatophagoides pteronyssinus),is the most important allergen.IgE antibody specifically binding to Der p 1 can be detected in serum of over 80%allergic organism.The allergy detection kit based on Der p 1 protein can be used for allergy specific diagnosis.At present,the allergy detection kit in China is mainly depends on imports,so it is urgent to establish an efficient production system of recombinant dust mite allergen to replace imported raw materials.In this study,recombinant expression of Der p 1 was performed based on Pichia pastoris expression system.First,after codon optimization,the full-length coding gene of PreProDer p 1 was expressed in Pichia pastoris GS115,and the yield was up to 100 mg/L.Further co-expression of molecular chaperones increased the yield to 140 mg/L.And the fermentation yield of 3 L reactor was increased to 1 g/L,which was the reported highest level.Afterwards,the purification process of the recombinant protein by cation exchange chromatography and affinity chromatography was optimized,and realized the purpose protein yield reached 60.7%.Finally,the bioactivity analysis showed that the purified protein had lower antigenic activity than the commercial product.The molecular weight analysis of the protein suggested that the cleavage of PreProDer p 1 might be uneven during the secretion,and the cleavage conditions of the leader peptide requires subsequent optimization.
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