Schmidt M, Zargari A, Holt P, Lindbom L, Hellman U, Whitley P, van der Ploeg I, Härfast B, Scheynius A
Department of Laboratory Medicine, Karolinska Hospital, Stockholm, Sweden.
Eur J Biochem. 1997 May 15;246(1):181-5. doi: 10.1111/j.1432-1033.1997.00181.x.
For the first time the complete cDNA encoding a major allergen and novel protein of the yeast Malassezia furfur, Mal f 1, has been sequenced and expressed. The amino acid sequences of nine tryptic peptides of the protein were determined. Oligonucleotides were designed from these amino acid sequences. The cDNA sequence was obtained by hybridizing these primers to mRNA and enhancement by reverse-transcriptase PCR techniques. The cDNA is 1176 bp in length. It shows an open reading frame of 1050 bp coding for a protein of 38178 Da and a deduced amino acid sequence containing 350 residues. The hydropathy plot and the tryptic digest indicate that the first 22 amino acids represent a leader sequence determining a mature protein of 35 988 Da. The complete encoding cDNA was expressed as a maltose-binding protein fusion protein in Escherichia coli. The recombinant fusion protein reacted with our specific monoclonal antibody and with IgE from patients with atopic dermatitis.
首次对编码酵母糠秕马拉色菌主要变应原和新蛋白Mal f 1的完整cDNA进行了测序和表达。测定了该蛋白九条胰蛋白酶肽段的氨基酸序列。根据这些氨基酸序列设计了寡核苷酸。通过将这些引物与mRNA杂交并利用逆转录酶PCR技术进行扩增，获得了cDNA序列。该cDNA长度为1176 bp。它显示了一个1050 bp的开放阅读框，编码一个38178 Da的蛋白，推导的氨基酸序列包含350个残基。亲水性图谱和胰蛋白酶消化表明，前22个氨基酸代表一个前导序列，决定了一个35988 Da的成熟蛋白。完整的编码cDNA在大肠杆菌中作为麦芽糖结合蛋白融合蛋白表达。重组融合蛋白与我们的特异性单克隆抗体以及特应性皮炎患者的IgE发生反应。
