粉尘螨主要变应原Derf 1基因的改造与可溶性表达

Prokaryotic Expression of Der f 1,A Major Allergen from the Dust Mite Dermatophagoides farinae

目的在大肠杆菌中提高粉尘螨1类变应原(Der f 1)的可溶性表达。方法用RT-PCR方法扩增得到Der f 1的全长序列与成熟肽序列(mDer f 1);以已知Der p 5基因的前导序列替换Der f 1前导序列和原酶序列,重新构建出rDer f 1基因;将上述3个基因分别克隆入原核表达载体pGEX-4T-1中表达,经Western-blot对三者的表达产物进行分析鉴定。结果Western-blot表明,pGEX-Der f 1与pGEX-mDer f 1的表达产物分别为分子量约63 000与51 000的重组蛋白质,重组蛋白质主要存在于细胞裂解液的沉淀中。pGEX-rDer f 1大量表达了可溶性目的蛋白质,分子量约53 000,并被成功地分离纯化。以上三种表达产物均可被鼠抗GST抗体与螨过敏患者血清特异地识别。结论表达了含Der f 1,mDer f 1与rDer f 1的三种GST融合蛋白质,它们均具免疫反应性,纯化获得了GST-rDer f 1融合蛋白质,为后期的诊断及治疗奠定了基础。

Objective To improve the prokaryotic expression of a major allergen Der f1 of Dermatophagoides farinae. Method Three Der f1 related sequences, namely the whole Der f1 gene, the mature enzyme sequence of Der f 1 （mDer f 1） and a recombinant Der f1 （rDer f1） containing a leader sequence from Der p 5 and the proenzyme sequence of Der f1, were obtained by RT-PCR. The amplified sequences were then cloned to vector pGEX-4T-1 and transformed into E. coli for protein expression. The target sequences were induced to express by IPTG and the recombinant proteins were then analyzed by Western-blot. rDer f1, the construct with a high expression level, was selected for further protein expression and purification studies. Result All the three target proteins can be detected by anti-GST antibody and patient serum. The two recombinant proteins, GST-Der f1 （mol. wt. 63 000 Mr） and GST-mDer f1 （mol. wt. 51 000 Mr）, were found to be expressed and remained inside E. coli. In contrast, a high level of soluble GST-rDer f1 （mol. wt. 53 000 Mr） was detected in the supernatant of the cell culture medium. GST-rDer f1 was also purified successfully. ConcIusion Der f1, mDer f1 and rDer f1 were expressed and the expressed proteins were found to react with anti-GST antibody and patient serum. Purified rDer f1 may be used for the future diagnosis and therapy of mite allergy.