The present study was aimed to produce the recombinant protein of Tyrophagus putrescentiae allergen component 32 (Tyr p 32) and to identify its immunoreactivity. The cDNA encoding Tyr p 32 was amplified from total RNA of T. putrescentiae and inserted into pET-28a (+) vector. The constructed plasmid pET-28a (+)-Tyr p 32 was transformed into BL21 (DE3) receptor cells. After being induced with IPTG, the recombinant protein was purified with Ni column, and then identified on SDS-PAGE and Western blotting. Serum of children with allergic asthma and(or) rhinitis was collected, IgE-ELISA and IgE-Western blotting were used to detect the binding rate of rTyr p 32 to human T.putrescentiae-positive serum. After human bronchial epithelial cells BEAS-2B being cultured with rTyr p 32, the expression levels of IL-6 and IL-8 cytokines was detected by ELISA and qRT-PCR, t-test was used for pairwise comparison between groups. The results showed that the cDNA length of Tyr p 32 was 885 bp. The sequence identity between Tyr p 32 and Der p 32, Der f 32 was 70.21% and 68.03%, respectively. According to SDS-PAGE and Western blotting, the molecular weight of rTyr p 32 was about 35 000 Da, which was consistent with the theoretical value. IgE-ELISA results showed that the positive rate of rTyr p 32 was 41.38% (12/29) against T. putrescentiae-positive serum. When BEAS-2B cells were cultured with rTyr p 32, the expression of IL-6 and IL-8 increased in the cell supernatant in a dose-dependent manner (t=-29.10,P=0.001 2;t=-33.69,P=0.000 9), which also significantly increased the mRNA expression levels of IL-6 and IL-8 (t=-9.15,P=0.011 7;t=-17.16,P=0.003 4). In conclusion, the recombinant protein rTyr p 32 was successfully prepared, which provides raw materials for component diagnosis and specific immunotherapy of allergic diseases.
制备腐食酪螨过敏原第32组分（Tyr p 32）重组蛋白并鉴定其免疫反应性。从腐食酪螨总RNA扩增Tyr p 32的编码基因，插入pET-28a（+）载体中，将pET-28a（+）-Tyr p 32质粒转化至BL21（DE3）感受态细胞中，经IPTG诱导表达后，用Ni柱进行层析纯化，用SDS-PAGE与Western blotting鉴定。收集过敏性哮喘和（或）鼻炎患儿血清，IgE-ELISA和IgE-Western blotting检测rTyr p 32与人腐食酪螨阳性血清结合率。将rTyr p 32与人支气管上皮细胞BEAS-2B共培养后，ELISA和qRT-PCR检测IL-6和IL-8细胞因子蛋白和mRNA表达水平，组间两两比较采用t检验。结果显示，获得Tyr p 32编码基因，全长为885 bp，Tyr p 32与Der p 32、Der f 32的氨基酸序列一致性分别为 70.21%和68.03%。SDS-PAGE和Western blotting结果显示rTyr p 32相对分子质量约为35 000 Da，与理论值一致。IgE-ELISA结果显示rTyr p 32与人腐食酪螨阳性血清的IgE结合率为41.38%（12/29）。当rTyr p 32与BEAS-2B细胞共培养后，rTyr p 32以浓度依赖性方式促进细胞上清中IL-6和IL-8的蛋白表达（t=-29.10，P=0.001 2；t=-33.69，P=0.000 9），并显著提高IL-6和IL-8 mRNA水平表达（t=-9.15，P=0.011 7；t=-17.16，P=0.003 4）。综上，成功制备具有免疫反应性的腐食酪螨过敏原重组蛋白rTyr p 32，为过敏性疾病组分诊断、特异性免疫治疗等提供了原料。.