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.
Production of recombinant protein of Tyr p 32 from Tyrophagus putrescentiae and identifying its immunoreactivity
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.
Tyrophagus putrescentiaeRecombinant proteinTyr p 32IgE binding rateImmunoreactivity
万方数据
维普
