Wagner B, Krebitz M, Buck D, Niggemann B, Yeang H Y, Han K H, Scheiner O, Breiteneder H
Department of General and Experimental Pathology, University of Vienna, Austria.
J Allergy Clin Immunol. 1999 Nov;104(5):1084-92. doi: 10.1016/s0091-6749(99)70093-x.
Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy.
The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy.
A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen.
An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3.
rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.
在脊柱裂（SB）相关的乳胶过敏中已发现两种天然橡胶乳胶蛋白，即Hev b 1和Hev b 3。
本研究旨在克隆并表达Hev b 3，获得具有免疫活性的可溶性重组变应原，用于诊断SB相关的乳胶过敏。
从马来西亚橡胶树（巴西橡胶树）采集的新鲜乳胶RNA中扩增编码Hev b 3的互补DNA（cDNA）。根据天然（n）Hev b 3内部肽段序列设计PCR引物。通过cDNA末端特异性扩增获得5′端序列。重组（r）Hev b 3在大肠杆菌中作为6xHis标记蛋白产生。进行免疫印迹和抑制试验以鉴定重组变应原。
获得了一个922 bp的Hev b 3 cDNA克隆，其编码一个由204个氨基酸残基组成的蛋白质，分子量为22.3 kd。在免疫印迹中，35例SB乳胶过敏患者中有29例显示IgE与rHev b 3结合，15例乳胶致敏组中有4例也显示IgE与rHev b 3结合。rHev b 3能够消除所有IgE与nHev b 3的结合，表明rHev b 3上存在所有IgE表位。Hev b 3与Hev b 1的序列同一性为47%。rHev b 3对IgE与nHev b 1结合的大量抑制说明了这两种乳胶变应原之间的交叉反应性。
rHev b 3是诊断SB患者乳胶过敏的合适体外试剂。Hev b 3全序列的确定和重组变应原的产生将有助于表位定位并改进乳胶过敏的诊断试剂。
