House dust mite allergen Der f enhanced bronchial epithelial cell cytokine expression

The house dust mites (Dermatophagoides farinae, Derf) are the major source of aeroaller gens implicated in the expression of atopic disorders, including asthma, allergic rhinitis and atopic dermatitis. In particular, strong circumstantial evidence suggests that house dust mite antigens are important precipitating factors of asthma. Many house dust mite allergens are proteases that can elicit airway inflammation by stimulating the release of cytokines from bronchial epithelial cells. To investigate whether Der f allergen proteases induced cytokine production from the epithelial cell line BEAS-2B, BEAS-2B cells were cultured with 4 different concentrations of Derf (0.02, 0.2, 2, 20μg/ml) for 24-96 h, after which supernatants were assayed for interleukin (IL)-6 and IL-8 with ELISA. Reverse transcription-PCR was also performed. The cell sheets were intact throughout the observation in control group without any exposure to Der f antigen. In the experimental groups cells treated with Der f allergen showed changes in the anchorage status of the monolayer. There was a significant increase in the level of cytokine production compared with the untreated sample. The release of IL-6 and IL-8 increased in a concentration-dependent manner (P <0.05, respectively) with the addition of increasing dosage of Der f to the cell sheets. Levels of IL-6 and IL-8 began to rise at 24 h and 48 h after allergen exposure, and they increased significantly in the supematants at 72 h and 96 h. At the same time the concentration dependence of induction of IL-6 and IL-8 expression as well as an increase in the expression of IL-6 and IL-8 mRNA manifested evidently. HDM-induced airway inflammation may include Der f-mediated release of inflammatory mediators, and the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium. It is suggested that IL-6 and IL-8 production by bronchial epithelial cells may play a role in the pathogenesis of allergic asthma.

粉尘螨变应原第6组分基因克隆及序列多态性分析

目的获得粉尘螨变应原第6组分(Der f 6)的编码基因并了解其序列多态性。方法根据GenBank(AF125187)公布的Der f 6的CDS区序列设计引物,使用PrimeSTAR?HS DNA进行PCR扩增,将目的基因片段与pMD19-T simple连接,进行序列测定和多态性分析。结果获得粉尘螨Der f 6编码基因,大小为839bp。对5个Der f 6克隆质粒测序并与参考序列GenBank No.AF 125187和GenBank No.EU085359比对,出现突变的cDNA序列合计36处,cDNA克隆中序列存在27处突变至少达到3个;推导的氨基酸序列有15处与上述27个位点一致。结论获得粉尘螨Der f 6编码基因并证明其序列具有多态性,为相关研究奠定了基础。

粉尘螨1类变应原T和B细胞表位t合基因的构建与表达

目的：构建粉尘螨1类变应原（Derf1）的T和B细胞表位嵌合基因原核表达载体。方法将Derf1包含的5个T细胞表位（T1～T5）和6个B细胞表位（B1～B6），以B1-T1-B2-T2-B3-T3-B4-T4-B5-T5-B6和B1-B2-B3-B4-B5-B6-T1-T2-T3-T4-T5连接方式直接合成表位嵌合基因，并分别命名为Derf1A和Derf1B。构建原核表达重组质粒pET-28a（＋）-Derf1A和pET-28a（＋）-Derf1B，双酶切及测序验证的阳性克隆转化到E.coliBL21（DE3）后诱导表达。SDS-PAGE分析表达产物后进行纯化，并进行Westernblotting鉴定。ELISA法检测嵌合蛋白对粉尘螨过敏患者血清IgE抗体的结合力。结果双酶切及测序结果表明，成功构建了原核表达重组质粒pET-28a（＋）-Derf1A和pET-28a（＋）-Derf1B；SDS-PAGE分析表明，Derf1A和Derf1B诱导并纯化成功，Westernblotting结果进一步证实纯化了Derf1A和Derf1B嵌合蛋白。与Derf1相比，Derf1A和Derf1B嵌合蛋白与粉尘螨过敏患者血清IgE抗体的结合能力显著降低（P均＜0.05），但Derf1A和Derf1B间差异无统计学意义（P＞0.05）。结论成功表达了Derf1的T和B细胞表位嵌合蛋白，为粉尘螨过敏的特异性免疫治疗奠定了基础。

粉尘螨3类变应原的T细胞表位预测及鉴定

目的预测并鉴定粉尘螨3类变应原(Der f 3)的T细胞表位。方法运用生物信息学分析软件预测Der f 3的T细胞抗原表位,并人工合成所预测的T细胞表位肽。采用改良MTT法,用预测表位肽刺激致敏鼠脾淋巴细胞进行脾淋巴细胞增殖试验;酶联免疫吸附试验检测脾细胞培养上清液中白细胞介素-2(IL-2)、γ干扰素(IFN-γ)、IL-4及IL-5水平。结果成功预测了Der f 3的5个T细胞表位肽,通过脾淋巴细胞增殖试验,其中3个表位肽序列可促进脾淋巴细胞增殖并刺激细胞因子IL-2和IFN-γ的分泌,抑制细胞因子IL-4和IL-5的分泌。其序列分别为37GDCPYQISLQSSSHFCGG54、98IYQHENYDSMTIDNDVALIKLKTPMT123和164SELQRVDIDVVSREQCDQLYS184。结论初步鉴定了Der f 3变应原中3个T细胞表位序列,为后续过敏性哮喘的诊断和特异性免疫治疗奠定基础。

粉尘螨3类变应原的B细胞线性表位预测及鉴定

目的预测并鉴定粉尘螨3类变应原(Der f 3)中的B细胞线性表位。方法运用生物信息学技术,从抗原性、表面易接近性、柔韧性、亲水性、β转角等5个方面在线预测Der f3的B细胞线性抗原表位。同时,采用人工合成法合成相应B细胞表位肽分别与由12份螨性过敏性哮喘患者血清组成的3个血清池共同作用,明确其中的强阳性表位肽序列。结果成功预测了Der f 3中的8个B细胞表位肽。其中,在过敏性患者血清与变应原特异性IgE结合试验中确认了33KAKAGDCP40、86HASGGEKIQVAEIYQHENYDSMTID110、118LKTPMTLDQTNAKPVPLPPQGSDVKVG144、199DVANGGVDSCQGDSGGPVVD218和156QEGSYSLP1635个强阳性表位肽序列。结论成功确认了Der f 3变应原中5个B细胞线性表位序列,为尘螨过敏性哮喘的诊断和治疗奠定基础。