抗粉尘螨主要变应原Der f1单克隆抗体的制备与鉴定

目的制备和鉴定鼠抗粉尘螨主要变应原Derf1的单克隆抗体（monoclonal antibody,McAb）。方法利用重组Derf1蛋白为免疫原,免疫BALB／c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。通过间接ELISA法筛选特异性分泌的杂交瘤细胞。用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行抗体纯化。采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型;通过间接ELISA、Western Blotting鉴定该单克隆抗体的特性和交叉性。结果获得6株可稳定分泌鼠抗粉尘螨主要变应原Derf1的单克隆抗体,其Ig亚型均为IgG1,且6株单抗均具有良好的效价。ELISA和Western—blotting分析表明,该6株单抗均能识别重组Derf1蛋白和天然的粉尘螨提取物。其中,除了484不识别屋尘螨提取物,其他均能识别。结论成功制备了6株鼠抗粉尘螨主要变应原Derf1的单克隆抗体,为建立粉尘螨主要变应原Derf1检测及纯化方法奠定了基础。     

屋尘螨变应原Der p 1基因原核表达产物的纯化及特性鉴定

目的建立屋尘螨主要变应原Derp1蛋白原核表达产物的纯化方法,获得理想的表达产物并鉴定其免疫原性。方法大量诱导表达含pET24a-Derp1质粒的BL21工程菌,表达产物以重组蛋白包涵体的形式存在,包涵体经洗涤与溶解后,使用结合6组氨酸的镍柱进行亲和层析纯化蛋白质,用稀释复性方法进行重组蛋白的复性,再用屋尘螨过敏性哮喘患者阳性血清经Dot-ELISA方法分析Derp1纯化蛋白的抗原活性。结果分步洗涤可有效去除重组蛋白包涵体沉淀中混杂的多数杂蛋白成分,亲和层析分离可获得高纯度重组变应原Derp1蛋白。Dot-ELISA法检测结果表明经复性并纯化的Derp1蛋白呈强阳性反应,最佳血清稀释度为1∶64,而重组蛋白与正常人血清呈阴性反应。结论纯化后的融合蛋白Derp1具有较高的纯度及较强的免疫活性,可望作为有效的屋尘螨变应原诊断试剂和疫苗的候选分子。     

粉尘螨Ⅰ类变应原成熟肽段基因的克隆和生物信息学Ⅰ分析

目的了解不同地区粉尘螨Ⅰ类变应原（DerfⅠ）成熟肽段基因序列的变异性及其编码蛋白的化学及免疫学特征。方法从皖南地区分离鉴定的活粉尘螨，提取总RNA，采用RT—PCR扩增DerfⅠ成熟肽段基因，克隆到T载体测序确认。应用生物信息学软件对该基因进行初步分析。结果获得皖南地区两种不同基因型的DerfⅠ成熟肽段基因，与GenBank公布的其他地区DerfⅠ基因序列比较同源性在98．73％～99．84％之间；相应编码氨基酸序列的同源性在99．04％～100％之间，存在6个氨基酸残基变异位点，并影响其抗原表位的改变。结论不同地区DerfⅠ成熟肽段基因序列及其编码氨基酸序列存在差异，并对其免疫原性产生影响。克隆并分析研究我国各地的尘螨变应原基因序列及氨基酸序列变化．对于临床变应性疾病的诊治有重要的意义。     

济南市东部过敏性呼吸道疾病患儿吸入性变应原谱分析

目的 调查济南东部地区过敏性呼吸道疾病患儿主要吸入性变应原.方法 采用20种标准化变应原对济南地区368例变应性呼吸系统疾病患儿进行皮肤点刺试验.并将样本中126例2～6岁的患者设为学龄前组,242例6～13岁的患者设为学龄组.比较两组在变应原阳性率分布上的差异.结果 变应原皮肤实验阳性率前8位的变应原分别为屋尘螨(69.6%)、粉尘螨(60.8%)、链格孢霉(23.1%)、艾蒿(20.2%)、刺槐(16.4%)、狗上皮(16.3%) 、烟曲霉(15.2%)、白色念珠菌(12.5%).学龄组中对粉尘螨和屋尘螨的敏感程度明显高于学龄前组(P<0.01).结论 尘螨、春秋季花粉及真菌是济南东部地区最重要的吸入性变应原.     

儿童支气管哮喘常见变应原调查

①目的了解青岛地区支气管哮喘儿童常见吸入性和食物性变应原的情况。②方法采用皮肤点刺法,对1454例支气管哮喘儿童进行变应原检测。其中吸入性变应原16种,食物性变应原8种。③结果吸入性变应原中粉尘螨和屋尘螨的阳性与强阳性反应率较高,分别为57．1％和62．1％,其中有23．2％的病儿仅表现为粉尘螨和屋尘螨的强阳性反应。食物性变应原中以海虾、蚌类、花生的阳性与强阳性反应率较高,分别为15．2％、24．1％和16．4％。④结论青岛地区诱发儿童支气管哮喘的主要吸入性变应原为粉尘螨和屋尘螨,食物性变应原主要为海虾、蚌类、花生。     

南宁市门诊变应性鼻炎患者的变应原谱分析

目的:分析我院门诊不同年龄组变应性鼻炎患者的主要变应原分布特点。方法:采用14种变应原对515例变应性鼻炎患者进行皮内试验,筛选致敏变应原,将样本中178例6～18岁的患者设为青少年组,337例19～65岁设为成人组,比较两组在变应原阳性率分布上的差异。结果:515例变应性鼻炎患者中,变应原阳性率按排列顺序青少年组前8位的变应原为:屋尘螨(94.38%)、粉尘螨(93.26%)、海鱼(78.09%)、海虾(78.09%)、夏秋花粉Ⅰ(69.67%)、春季花粉Ⅰ(67.42%)、桑蚕丝(61.80%)、棉絮(57.31%);成人组前8位的变应原为:屋尘螨(87.24%)、粉尘螨(86.94%)、海鱼(86.65%)、海虾(81.60%)、棉絮(70.62%)、夏秋花粉Ⅰ(63.80%)、桑蚕丝(61.72%)、春季花粉Ⅰ(58.16%)。结论:南宁市无论是青少年组还是成人组,粉尘螨及屋尘螨变应原阳性率高居首位,其次是海鱼、海虾食物性变应原。     

420例维吾尔族变应性鼻炎患儿变应原测试和分析

目的:分析维吾尔族儿童变应性鼻炎变应原的常见种类及分布特点。方法:对420例前来我院就诊的维吾尔族变应性鼻炎患儿进行变应原皮肤点刺试验(SPT)。将患儿按年龄分为婴幼儿组、学龄前组和学龄组,比较各组SPT及各变应原阳性率结果:患儿主要变应原为粉尘螨和屋尘螨,阳性率分别为67.8%和65.4%。各年龄组变应性鼻炎患儿SPT阳性率比较,差异有统计学意义(P<0.05)。结论:维吾尔族变应性鼻炎患儿的主要过敏原为屋尘螨和粉尘螨;变应性鼻炎患儿SPT阳性率与年龄有一定相关性。     

吸入性变应原检测在吉林地区变应性鼻炎患者中的应用

目的探讨引起吉林地区变应性鼻炎(AR)的主要吸入性变应原,为临床上AR患者的诊断及治疗提供客观依据。方法以艾蒿花粉、豚草花粉、螨虫组合(屋尘螨/粉尘螨)、树木组合(柳树/杨树/榆树)、屋尘、猫上皮、狗上皮、蟑螂、点青霉/分支孢霉/烟曲霉/交链孢霉、啤酒花作为变应原,将样本中6～18岁患者设为儿童组70例,19岁以上患者设为成人组140例。采用欧蒙印迹法检测210例AR患者血清中变应原特异性IgE抗体,并对结果进行相应性分析。结果 210例AR患者中,检测阳性率居于首位的是艾蒿花粉,其次为树木组合(柳树/杨树/榆树)、螨虫组合(屋尘螨/粉尘螨)、屋尘、豚草花粉、猫上皮、狗上皮、蟑螂、点青霉/分支孢霉/烟曲霉/交链孢霉、啤酒花。变应性鼻炎患者对食物过敏者比较少见。结论艾蒿、树木组合(柳树/杨树/榆树)、螨虫组合(屋尘螨/粉尘螨)和屋尘是吉林地区变应性鼻炎患者主要的吸入性变应原。     

南通地区变应性鼻炎合并哮喘患者吸入性变应原分析

目的调查分析南通地区变应性鼻炎合并哮喘患者主要吸人性变应原。方法采用16种标准化变应原对南通地区250例变应性鼻炎合并哮喘患者进行皮肤点刺试验（SPT）,分析变应原的分布特征,并比较未成年组与成人组变应原阳性率分布差异。结果南通地区变应性鼻炎合并哮喘患者主要吸人性变应原是粉尘螨（62．4％）、屋尘螨（58．8％）、蟑螂（15．6％）。未成年组对粉尘螨、屋尘螨的敏感程度均高于成人组（均P〈0．05）。结论尘螨是南通地区变应性鼻炎和哮喘患者最主要吸入性变应原。     

437例慢性荨麻疹患者皮肤点刺试验结果分析

目的 了解东莞地区慢性荨麻疹患者的常见变应原分布情况.方法 对进行皮肤点刺试验的 437例慢性荨麻疹患者做27种变应原测定,并对结果进行总结分析.结果 437例慢性荨麻疹患者中食物组变应原阳性率前6种是金枪鱼(71.4%)、虾(43.2%)、芹菜(34.1%)、蚌(31.1%)、鲤鱼(29.3%)、牛奶(24.7%);吸入组变应原阳性率前6种是屋尘螨(67.3%)、粉尘螨(67.0%)、禾本科(花粉)(33.2%)、杂草(28.4%)、禾本科(谷类)(20.1%)、云杉(19.0%).其中金枪鱼、虾、屋尘螨和粉尘螨阳性率均在40%以上,是东莞地区慢性荨麻疹患者的主要变应原.25种变应原阳性率比较与年龄无关,2种(粉尘螨和云杉)变应原阳性率比较与年龄有关.结论 金枪鱼、虾、屋尘螨和粉尘螨是东莞地区慢性荨麻疹的主要变应原,进行皮肤点刺试验可以有效寻找过敏原,减少病情反复机会.     

Allergen micro-array detection of specific IgE-reactivity in Chinese allergy patients

Background  Allergen micro-arrays are powerful tools for screening of serum IgE-reactivity. In this study allergen micro-arrays were used to identify dominating IgE-binding allergens and cross-reactivity patterns among selected Chinese allergy patients.

Methods  The study was conducted using patient sera from the cities of Guangzhou, Nanjing, Chengdu and Shenyang. In total 100 sera with Dermatophagoides pteronyssinus (Der p) specific IgE-levels higher than 50 kU/L were selected for testing against 103 individual allergens.

Results  Among 100 selected patients, 95% showed IgE-reactivity towards house-dust mite allergens Dermatophagoides farinae (Der f) 1, Der f 2 and Der p 2 and 94% were IgE positive against Der p 1, and 60% of sera contained IgE reacting against allergen Euroglyphus maynei (Eur m) 2. IgE against cat allergen, Felisdomesticus (Fel d) 1, was seen in 20%. Only 2% showed specific IgE-reactivity to Der p 10, a panallergen belonging to the tropomyosin family. Serum IgE-reactivity towards other allergens was in general low. IgE-reactivity against pollen allergens showed geographic differences.

Conclusions  This study clearly confirms that group 1 and group 2 are major allergens of house dust mites. These selected house-dust mite allergy patients are close to being mono-sensitized. Der p 10 is not an important allergen for cross-reactivity. Specific IgE-sensitization towards pollen allergens is low in southern China compared to other regions. The prevalence of food and stinging insect allergens known to give rise to IgE-mediated cross-reactivity is 2% or less.

     

Indoor Allergen Levels and Household Distributions in Nine Cities Across China

Objective Chinese allergic subjects have high levels of sensitization to house dust mite (HDM) and other indoor allergens. This study quantifies common indoor allergen levels in Chinese households.
Methods Dust samples were collected from nine cities. Major allergens Der p 1 and Der f 1 from Dermatophagoides pteronyssinus and D. farinae, and specific antigens of Blomia tropicalis, Tyrophagus putrescentiae, Acarus siro, and cockroach species Blattella germanica and Periplaneta americana were measured by ELISA.
Results HDM allergens were found in dust samples from bedding in 95% of the Chinese households. The median levels varied from <0.006 to 9.2 μg/g of dust, depending on the city. The percentages of households having HDM allergen levels associated with the risk of developing allergy sensitization and asthma were 65% and 25%, respectively. Specific antigens of the storage mite and cockroach were only found in samples from the southern and tropical regions of China. Levels of mite allergens were generally higher in samples from bedding compared to samples from the living room, even for storage mites, whereas levels of cockroach antigens were higher in the living room samples.
Conclusion HDM allergens are present in bedding dust samples from most Chinese households. Cities in southern and central China have relatively high levels of HDM major allergens compared to cities in northern and western China. Antigens of storage mites and cockroaches are not as common as HDM allergens.     

Comparison of Three Methods of Protein Extraction from Dermatophagoides Pteronyssinus for Two-dimensional Electrophoresis

Obiective To explore an effective method of Dermatophagoides pteronyssinus protein extraction suitable for two-dimensional electrophoresis （2-DE） analysis. Methods The extracts of Dermatophagoides pteronyssinus were prepared with Coca＇s solution, lysis buffer of 2-DE, and Trizol reagent, respectively. Bicinchoninic acid （BCA） assay was used to determine the total protein concentration of the samples. The efficiency of different protein extraction methods were evaluated with 2-DE analysis. Results The concentrations of extracted protein by methods of Coca＇s solution, lysis buffer, and Trizol reagent were 0.63 g/L, 0.90 g/L, and 0.80 g/L, respectively. The 2-DE analysis results showed that some protein spots in low molecular weight （LMW） range could be detected with the Coca＇s solution method. With the lysis buffer of 2-DE method, more protein spots in LMW range could be detected, while the medium molecular weight （MMW） protein spots were absent. Several MMW protein spots （174-178 kD and 133 kD） and more LMW protein spots were detected withTrizol reagent method. Conclusions Among Coca＇s solution, lysis buffer of 2-DE, and Trizol reagent, the concentration of extracted protein of Dermatophagoides pteronyssinus by lysis buffer of 2-DE is the highest. However, most protein components of Dermatophagoides pteronyssinus purified mite bodies can be extracted by Trizol reagent, which may generally reflect the whole profile of Dermatophagoides pteronyssinus allergens.     

Cloning, sequence analysis and expression in E. coli of the group 3 allergen of Dermatophagoides farinae

Background The dust mites, which are mostly represented by Dermatophagoides spp. （Acari： Pyroglyphidae）, are the major sources of indoor allergens. Identification and characterization of these mite allergen molecules are an important step in the development of new effective diagnostic procedures and possible therapeutic strategies for allergic disorders associated with dust mites. Methods Total RNA was extracted from Dermatophagoides farinae. The gene coding for Der f 3 was amplified by RT-PCR with the primers designed based on previous sequence published in GenBank. The target gene was cloned intermediately into pMD19-T plasmid and finally into plasmid pET28a （＋）, expressed in E. coil BL21 at the aid of the inducer isopropyI-D-thiogalactopyranoside （IPTG）. The physicochemical properties, spatial structure of the allergen were analyzed with bioinformatics software. Results The cDNA coding for group 3 allergen of Dermatophagoides farinae from China was cloned and expressed successfully. Sequencing analysis showed that there were nineteen mismatched nucleotides in five Der f3 cDNA clones in comparison with the reference （GenBank Accession No. AY283291）, which resulted in deduced amino acid sequence incompatibility in eleven residues. Bioinformatics analysis revealed that the Der f 3 pro-protein was an extracellular hydrophobic protein, consisting of 259 amino acids with a 16 amino acid signal peptide. The protein was deduced to have three chymotrypsin active sites （53-68 AA, 108-122 AA and 205-217 AA）, one N-glycosylation site, one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, and five N-myristoylation sites. Conclusions Der f3 is an extracellular hydrophobic protein which possesses multiple activation and phosphorylation sites. Polymorphism may exist in the Der f3 gene but this needs to be further confirmed in the future.     

粉尘螨的培养实验

目的:评价应用配合饲料培养粉尘螨的效果。方法:在粉尘螨培养环境相同的条件下(温度25℃±1℃,相对湿度80%±1%),应用配合饲料(A组)和纯面粉(B组)分别培养粉尘螨,经过一定时间的饲养,对子代进行计数。结果:通过对两组子代数量进行分析,A组明显多于B组,差异极显著(P<0.01)。结论:在粉尘螨的培养中,选用配合饲料作为培养料,可以在一定的时间内培养出大量的粉尘螨,为研究螨性疫苗制剂提供原材料。     

粉尘螨的生境与季节消长

粉尘螨是人体螨病的主要病原螨之一。我们自１９９５年１２月至１９９６年１１月对该螨的生境及季节消长进行了研究。结果显示,粉尘螨生境分布广泛,在地脚粉,动物饲料及部分中药材中孳生密度较高。孳生密度自５月份起增高,至７、８月份达到高峰,１０月份开始下降,全年可维持５个月的较高水平。提示,有关职业人员在此期间应做好防护工作。     

粉尘螨种群消长及空间分布型研究

目的初步调查粉尘螨在学生宿舍中的种群数量消长及空间分布型研究。方法每月5、15、25日定点采集螨样本,鉴定及计数。采用扩散型指标、Taylor冥法则及Iwao m*x回归分析法对数据进行分析。结果粉尘螨数量高峰期出现在6月和9月,各扩散型指标为I>0,CA>0,m*/x>1;Taylor幂法则回归方程为lgS2=0.2912+1.2721lg x;Iwao回归式为m*=0.6672+1.082x。结论粉尘螨在学生宿舍中种群消长曲线呈双峰型,其空间分布格局呈聚集分布。     

Comparison and Evaluation of Several Dermatophagoides Pteronyssinus Allergen Extracts for Skin Prick Test

Objective To evaluate the significance of several Dermatophagoides pteronyssinus allergen extracts for skin prick test （SPT） in patients allergic to Dermatophagoides pteronyssinus. Methods Two hundred and nineteen patients enrolled in Peking Union Medical College Hospital underwent SPT and serum specific IgE assay to detect the Dermatophagoides pteronyssinus allergen. Three kinds of house dust mite allergen extracts were used for SPT, including the Dermatophagoides pteronyssinus extract prepared by our laboratory （group A）, standardized Dermatophagoides pteronyssinus extract （group B）, and mixed extracts of Dermatophagoides pteronyssinus and Dermatophagoidesfarinae （group C）. Human serum specific IgE result was regarded as the reference standard for diagnosis of Dermatophagoides pteronyssinus allergy. The receiver operating characteristic （ROC） curve was used to evaluate the diagnostic performance of SPT with the extracts of three groups.Results SPT results showed that the median wheal diameter of group A, group B, and group C was 0.43, 0.35, and 0.28 cm, respectively, with significant difference among three groups （P〈0.05）. The difference was significant between group A and B （P〈0.01） as well as group A and C （P〈0.01）, but not between group B and C （P〉0.05）. There was no local urticaria or systemic allergic reactions following the procedure of SPT. Local reaction was observed in 5 patients and delayed reaction was in 2 patients of group A. As for group B and C, local reaction occurred in 3 cases and delayed reaction in 2 cases in each group. The area under ROC curve of SPT with extract in group A, group B, and group C was 0.765, 0.801, and 0.782, respectively. Based on the detection results of serum specific IgE, the sensitivity of SPT in diagnosis of Dermatophagoides pteronyssinus allergy with extract of group A, group B, and group C was 92.4%, 87.0%, and 81.5%, and the specificity was 60.6%, 73.2%, and 74.8%, respectively. Conclusion The Dermatophagoides pteronyssinus extract for SPT prepared by our laboratory offers good sensitivity and specificity comparable to commercially available allergen extracts, and it may be an appropriate candidate for clinical screening and diagnosis of Dermatophagoides pteronyssinus allergy.     

屋尘螨第13组变应原克隆表达、纯化及免疫原性鉴定

目的 克隆表达及纯化屋尘螨（Dermatophagoides pteronyssinus, Der p）第13组变应原（Der p13）蛋白,并鉴定其免疫原性。方法 提取屋尘螨总RNA,通过逆转录聚合酶链式反应（RT-PCR）技术获得Der p13的cDNA片段。正确设计引物,利用已获得的cDNA片段进行PCR扩增,获得Der p13基因片段。将扩增产物连接到载体上并转入克隆菌Top10中,挑选阳性菌落,保菌取样送到上海生工生物有限公司进行测序。将测序正确的基因转入表达载体PET-24a中,经BamH I、Xho I双酶切后用异丙基硫代半乳糖苷（IPTG）诱导表达和镍柱亲和层析法纯化重组蛋白Der p13。使用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）以及免疫印迹（Western Blot）鉴定纯化产物。结果 成功构建表达了质粒PET-24a-Der p13,SDS-PAGE电泳结果显示上清可溶性蛋白表达量较多,分子质量约为13 000。免疫印迹结果表明该蛋白可与尘螨过敏患者血清结合,具有一定免疫原性,重组蛋白Der p13三级结构预测表明其主要是β折叠,根据DNAStar抗原指数、亲水性、表面可及性、柔韧性等数据可以推导出蛋白Der p13抗原区,重组蛋白Der p13进化树分析结果显示屋尘螨与粉尘螨、疥、害嗜鳞螨及储存螨同源性较高。结论 成功克隆表达、纯化出Der p13重组蛋白,该蛋白具有一定的免疫原性,为临床实验研究过敏性疾病的诊断与治疗奠定基础。     

粉尘螨变应原Derf1基因的原核表达及多克隆抗体的制备

目的:原核表达、纯化粉尘螨主要变应原Der f1重组蛋白,并制备其多克隆抗体。方法:大量诱导表达含pET-28a(+)-Der f1质粒的BL21菌株,SDS-PAGE电泳并割胶纯化目的蛋白,以此为抗原免疫新西兰大白兔,收集血清进行效价检测并用Western blot检测其特异性。结果:割胶回收可有效纯化Der f1重组蛋白,抗Der f1抗体效价为1∶32 000。结论:成功制备Der f1多克隆抗体,以期为尘螨过敏性疾病的诊断和免疫治疗提供参考。