艾蒿、青蒿花粉变应原组分的研究

目的 对艾蒿、青蒿花粉变应原进行分离、鉴定。方法 采用不同的提取方式得到艾蒿、青蒿花粉的粗浸液，通过饱和(NH4)2SO4分级沉淀、聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白质组分，并用凝胶成像系统测定各组分的相对分子质量(Mc)；采用Western-blotting鉴定2种花粉的主要及次要变应原。结果 艾蒿、青蒿花粉分别分离到二十和十多种蛋白质组分。其中艾蒿花粉的组分中有9种蛋白能与患者血清中蒿属花粉特异性IgE结合，Mr为62000、43000、38000的蛋白条带的结合率最高。青蒿花粉的组分中有11种蛋白能与患者血清中蒿属花粉特异性：[gE结合，肘。为43000、38000的蛋白条带结合率最高。结论 艾蒿花粉的主要变应原Mr分别为62000、43000和38000，青蒿花粉的主要变应原Mr分别为43000和38000；2种花粉变应原组分存在很大相似性，但也有各自特异的变应原组分。     

重阳木花粉过敏原的分离、纯化和鉴定

目的对重阳木花粉变应原蛋白进行分离、纯化和鉴定。方法采用Coca s液提取重阳木花粉的粗提液,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离粗提液蛋白质组分,并测定其分子质量;收集过敏患者血清,用Western blot法鉴定其变应原成分;通过离子交换层析对重阳木花粉变应原进行初步纯化和免疫印迹鉴定。结果分离得到重阳木花粉18条蛋白带,其中分子质量为12和14 ku的是重阳木花粉的特异性变应原,通过离子交换柱层析方法纯化得到其相应的纯化蛋白。结论对重阳木花粉变应原进行了初步的分离、纯化和鉴定,为临床重阳木花粉过敏疾病的诊断和治疗奠定了基础。     

短穗鱼尾葵花粉泛过敏原profilin基因的克隆表达、纯化及免疫学鉴定

目的克隆并表达短穗鱼尾葵花粉中泛变应原肌动蛋白抑制蛋白（profilin）。方法利用RT-PCR结合RACE技术克隆短穗鱼尾葵花粉中泛变应原profilin的全长基因,并进行序列分析。然后设计带有酶切位点的特异性引物,采用RT-PCR获得整个短穗鱼尾葵花粉profilin的开放阅读框,将其与pET28a载体连接并转化大肠杆菌BL21（DE3）进行诱导表达,通过Ni2＋亲和层析柱对重组蛋白进行纯化,采用Western-blot检测其IgE结合活性。结果克隆获得了短穗鱼尾葵花粉profilin的全长基因,由608个碱基组成,开放阅读框为396个碱基（包括终止密码子）,编码131个氨基酸。该序列编码的蛋白为小分子量酸性蛋白,等电点为4.52,相对分子质量约为14200。此序列已被GenBank收录,登陆号为EF173600。重组短穗鱼尾葵花粉profilin在大肠杆菌中高效的表达,进一步经Ni2＋亲和层析柱纯化后经Western-blot检测具有良好的免疫学活性。结论成功地克隆和表达了短穗鱼尾葵花粉profilin,为短穗鱼尾葵花粉过敏的诊断和免疫治疗奠定了基础。     

重阳木花粉过敏原的分析、鉴定与纯化

 【摘要】 目的　对重阳木花粉变应原蛋白进行分析、鉴定与纯化。方法　提取这重阳木花粉的粗提液,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS - PAGE)分离粗提液蛋白质组分并测定其分子量,收集过敏病人血清,采用免疫印迹(Western - blotting)法鉴定其变应原成分,通过离子交换层析对重阳木花粉变应原进行初步纯化和免疫印迹鉴定。结果　重阳木花粉有18条主要蛋白带,12 000Mr和14 000Mr为重阳木花粉特异性变应原;通过离子交换层析方法纯化出重阳木花粉分子量为12 000Mr和14 000Mr的变应原主要分布在II峰中。结论　对重阳木花粉变应原进行了初步的分离、鉴定和纯化,为临床重阳木花粉过敏疾病的诊断和治疗奠定了基础。     

王棕花粉变应原的分析与鉴定

目的对我国南方常见的棕榈科植物王棕花粉（Roystonea regia pollen）变应原蛋白进行分离、分析与鉴定,为标准化变应原疫苗的研制提供基础。方法取常规方法制备的王棕花粉浸出液,采用SDS．PAGE分离王棕花粉蛋白质组分,测定其相对分子量,同时用10例对王棕花粉过敏的患者血清作Western-blot鉴定其变应原及主要变应原成分。结果SDS．PAGE显示王棕花粉有10条可辨蛋白带,其中主要条带有8条,分别为100000、66000、38000、36000、29000、30000、24000、16000和14000Mr,Western—blot结果表明,10例王棕花粉过敏患者血清全部呈阳性反应,有66000、24000、16000和14000Mr共4条致敏条带,其中分子量在16000和14000Mr的蛋白为主要变应原。结论王棕花粉变应原的分析与鉴定为临床王棕花粉变态反应疾病的诊断和治疗奠定了基础。     

油菜花粉过敏原的分析、鉴定与纯化

目的对油菜花粉的变应原组分进行鉴定及初步的分离及纯化。方法提取油菜花粉粗提液,然后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离油菜花粉的蛋白质组分并测定其相对分子质量,采用免疫印迹(Western blotting)法鉴定其变应原成分,并通过离子交换层析对油菜花粉变应原进行初步分离纯化,免疫印迹进行检测。结果油菜花粉粗提液有10余条蛋白带,其中相对分子质量为30 000、25 000、15 000和10 000的蛋白可与油菜花粉过敏性病人血清IgE结合,其中15 000和10 000为主要变应原;离子交换层析结果显示主要过敏原成分主要分布在Ⅰ、Ⅱ和Ⅲ峰中。结论对油菜花粉变应原进行了初步的分离、鉴定和纯化,为临床油菜花粉变态反应疾病的诊断和治疗奠定了基础。     

德国小蠊变应原皮内试验与血清sIgE检测的相关性

目的探讨德国小蠊变应原皮内试验与血清sIgE检测的相关性。方法通过对德国小蠊变应原天然粗浸液进行酶联免疫吸附实验（ELISA）测定蟑螂过敏病人血清sIgE水平,与皮试结果进行相关性比较。结果当皮试阳性反应程度≥“＋”时,sIgE阳性率为41.7%,皮试与ELISA的符合率为65.6%;当皮试阳性反应程度≥“＋＋”时,sIgE阳性率为60%,皮试与ELISA的符合率为87.5%,皮试程度与血清sIgE抗体检测结果显著相关;当皮试阳性反应程度≥“＋＋＋”时,皮试与ELISA完全相符。结论强阳性皮试反应程度与ELISA检测血清sIgE水平呈一致关系,两种方法可相结合用于分析诊断德国小蠊变应原。     

椰子花粉过敏原的分离、鉴定与纯化

目的 对椰子花粉的变应原组分进行初步的分离、鉴定及纯化.方法 提取椰子花粉粗提液,用十二烷基硫酸钠.聚丙烯酰胺凝胶电泳(sDS-PAGE)分离椰子花粉的蛋白质组分并测定其相对分子质量,采用免疫印迹法鉴定其变应原成分,并通过离子交换层析对椰子花粉变应原进行初步分离纯化,免疫印迹进行检测.结果 SDS-PAGE显示椰子花粉粗提液有10条蛋白带,其中相对分子质量(肘,)为60 000、50 000、35 000、28 000、19 000、16 000和14 000的蛋白可与椰子花粉过敏性病人血清IgE结合,且M,50 000、16 000和14 000为主要变应原;离子交换层析结果显示主要过敏原成分主要分布在V峰中.结论 对椰子花粉变应原进行了初步的分离、鉴定和纯化,为临床椰子花粉变态反应疾病的诊断和治疗奠定了基础.     

紫红笛鲷过敏原的提取、分离及其免疫学特性鉴定

目的对紫红笛鲷过敏原进行提取、分离及免疫学特性鉴定。方法新鲜紫红笛鲷经预处理后用PBS缓冲液制备总蛋白粗浸液，SDS-PAGE分析紫红笛鲷总蛋白的组成，免疫印迹（Western-blotting）分析紫红笛鲷过敏原，通过离子交换层析对总蛋白粗浸液进行分离并鉴定不同组份的免疫学特性。结果紫红笛鲷可溶性蛋白粗提液SDS-PAGE显示有20条蛋白条带，对鱼过敏病人的阳性混合血清能与其中7个条带反应，分子量分别是42000，36000，30000，27000，25000，17000和12000Mr。离子交换层析后分子量为42000、36000、12000Mr的阳性过敏原蛋白具有免疫学活性。结论本实验对紫红笛鲷过敏原进行了提取、分离和免疫学特性鉴定，离子交换层析技术可以用于紫红笛鲷过敏原蛋白的分离纯化，为紫红笛鲷过敏原的进一步研究和鱼类食品过敏的防治奠定了理论基础。     

梭子蟹变应原的分离、纯化与鉴定

目的：对梭子蟹（Portunus pelogicus（Linnaeus））变应原进行分离,鉴定其主要及次要变应原,采用蛋白纯化技术获取梭子蟹天然的主要变应原并进行鉴定,为标准化变应原疫苗的研制提供理论依据.方法：取常规方法制备梭子蟹浸出液,经SDS-PAGE分离,测定各组分的相对分子量;同时用26例对蟹过敏的病人血清进行Western blot,鉴定其主要及次要变应原;利用快速制备液相色谱（FPLC）纯化技术（凝胶过滤层析和离子交换层析）获取主要变应原并作鉴定.结果：SDS-PAGE显示梭子蟹有19条可辨蛋白带,分子量在13 000～90 000之间,其中主带有9条,分子量是20 900、24 200、27 100、29 200、33 700、38 900、48 700、74 700、89 100;Western blot结果表明,26例蟹过敏患者血清全部呈阳性反应,浸出液中共有5条致敏条带,其中分子量在74 400、48 700的是主要变应原,阳性反应率均为100%;纯化后获取了74400、48700的主要变应原;经过免疫鉴定证实其具有免疫活性.结论：梭子蟹74 400和48 700的变应原为主要变应原,层析技术可以对分子量为74400和48700的主要变应原成分进行纯化.     

IgE crossreactivity between mugwort pollen (Artemisia vulgaris) and hazelnut (Abellana nux) in sera from patients with sensitivity to both extracts

Background An association between sensitization to Compositae pollens and hypersensitivity to hazelnut has been previously described. There is no previous in vitro study about crossreactivity between mugwort pollen and hazelnut. Objectives To study mugwort pollen and hazelnut allergens and to assess if there is IgE crossreactivity between mugwort pollen and hazelnut. Methods A serum pool formed by 28 individual sera with specific IgE to mugwort pollen and hazelnut was used to investigate IgE crossreactivity. RAST-inhibition, SDS-PAGE/IEF immunoblotting inhibition assays were performed by preineubation of the sera with mugwort pollen and hazelnut. Results RAST to hazelnut was inhibited up to 63% by mugwort pollen, but the mugwort pollen RAST was only inhibited up to 36% by hazelnut. In SDS-PAGE immunoblotting mugwort pollen showed nine allergens ranging from < 16 to 65kDa and hazelnut had four main allergens: 42kDa, 17kDa and < 16kDa (two bands). In the SDS-PAGE immunoblotting inhibition hazelnut partially inhibited all the mugwort pollen bands, except that with 19kDa, whereas mugwort pollen produced a nearly total inhibition of all the hazelnut allergens. In isoeleetrofocusing immunoblotting mugwort pollen had two groups of allergens: pI 7.5–8.5 and pi 3.5–5.2 and hazelnut one group of allergens: pI 5.2–5.8. In the isoeleetrofocusing immunoblotting inhibition hazelnut produced a partial inhibition of all the bands of mugwort pollen and mugwort pollen partially inhibited all the allergenic bands of hazelnut. Conclusions The RAST and SDS-PAGE/IEF immunoblotting inhibition results provide evidence of IgE cross reactivity between mugwort pollen and hazelnut allergens. The inhibition of hazelnut by mugwort pollen is higher than the inhibition of mugwort pollen by hazelnut in both RAST inhibition and SDS-PAGE immunoblotting inhibition. These results suggest that mugwort pollen allergens would behave as primary immunogens in the association between sensitivity to mugwort pollen and hazelnut.     

Heterogeneity of a major allergen from olive (Olea europea) pollen.

Studies were carried out in order to confirm and extend knowledge of the physico-chemical properties of an allergenic material found in the pollen of the olive tree (Olea europea). The sera from 88% of patients who were sensitive to olive pollen contained IgE that reacted with a 19,000 Mr component and many also reacted to a 17,000 Mr band as shown by SDS-PAGE immunoblotting. A monoclonal antibody (OL-1) produced to the 19,000 Mr component also reacted with the 17,000 Mr band, and with bands at 21,000 and 41,000 under non-reduced conditions. HPLC separation followed by SDS-PAGE and immunoblotting of the fractions indicated that the allergen fraction from which the 19,000 and 17,000 components were derived had a mol. wt between 50 and 60 kD. Isoelectricfocusing followed by immunoblotting and development with (OL-1) indicated heterogeneity of the allergen with respect to pI values. Two of the strongest components of the six identified which reacted with (OL-1) had pIs of about 5.0 and 6.0 confirming the published data. The study therefore showed that olive pollen contains a number of allergenic components, with various mol. wts and pI values, with some epitopes in common, which may in the native state be bound together or aggregated.     

Identification and characterization of important allergens from mugwort pollen by IEF, SDS-PAGE and immunoblotting

Mugwort (Artemisia vulgaris L.) pollen allergens, separated by SDS-PAGE or IEF, were identified after transfer to NCM by incubation with a panel of sera from 16 patients with clinical mugwort pollen allergy, followed by [125I]anti-IgE and autoradiography. Of the at least 23 components separated by SDS-PAGE in a 15% polyacrylamide gel, at least 15 components with mol. wts 12,000-100,000 bound IgE from the panel of patient sera. A component of mol. wt 22,000 bound IgE from at least 94% of the patient sera tested and for all but three sera this component also bound the greatest quantity of IgE. Five other components with mol. wts 12,000, 17,000, 29,000, 39,000 and 42,000 bound IgE from 75-94% of the patient sera. After separation by IEF, at least 28 protein bands were detected in the pI region 3.5-7.2 and at least seven bands were found in the region 8.6-9.3. At least 11 bands in the pI range 4.2-7.3 and at least five bands in the pI region 8.5-9.2 bound IgE from the panel of patient sera. The most intense radiostaining was observed with a component having a pI of 4.35, which bound IgE from 31% of the patient sera. Immunoblotting of the SDS-PAGE and IEF gels using specific rabbit antisera and human sera against three important mugwort pollen allergens, denoted Ag 9, Ag 12 and Ag 13, was performed to determine the mol. wt and pI of these allergens which had earlier only been identified in CIE/CRIE. The results revealed that Ag 13 had a mol. wt of 61,000 and a pI of 4.35, Ag 12 had a mol. wt of 22,000 and AG 9 had pIs in the region 4.55-5.55 (six isoforms). Ag 9 did not bind IgE after SDS-PAGE and was thus not identified in the SDS-PAGE pattern, and Ag 12 failed to be detected in the NCM after transfer from IEF gels. By crossed immunoelectrofocusing, Ag 12 was found to consist of several isoforms predominantly located in the pI region 3.5-5.1. The immunoblotting analysis also revealed that the glycoprotein allergen Art v II was not detected after transfer from either SDS-PAGE or IEF gels. In conclusion, immunoblotting analysis of SDS-PAGE and IEF gels are useful methods for characterization of mugwort pollen extract, but it should be noted that some important allergens which are easily identified in CIE/CRIE may fail to be detected by these methods.     

Identification and partial purification of pollen allergens from Artemisia princeps

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.     

Identification of orchard grass (Dactylis glomerata) pollen allergens following electrophoretic transfer to nitrocellulose

Orchard grass (cocksfoot) pollen extracts, fractionated by polyacrylamide gradient electrophoresis or SDS gel electrophoresis were electroblotted onto nitrocellulose membranes and probed with sera from orchard grass pollen-allergic patients and 125I-anti-human IgE. The IgE-binding components of the pollen were detected by autoradiography. Elution studies showed that allergens could be extracted immediately and continuously over a 3-hour period. Two fractions of MWs 28,000 and 30,000 could be detected only after 20 min extraction. SDS-PAGE separations gave the better resolution revealing 19 electrophoretically-separate components, 13 of which bound human IgE. All of the IgE-binding components had MWs in the range 14,000 - 70,000. Three of the bands bound IgE from more than 85% of the serum samples. Following gradient gel electrophoresis, IgE binding was exhibited by 10 bands in the range MW 5,000 to greater than 669,000. The technique used allows one to quantitatively examine patients' sera for allergen-specific IgE antibodies and to identify the clinically important allergens. Results revealed numerous allergenic components over a wide MW range while patterns of IgE binding with different patients' sera demonstrated a great diversity of IgE antibody responses. This study demonstrates the suitability of the electroblotting technique combined with autoradiography for the investigation of allergenic components of grass pollen extracts and hence has application to extract standardization and immunotherapy. Such studies can be carried out rapidly, economically and with a high degree of sensitivity.     

Occupational rhinitis and bronchial asthma due to artichoke (Cynara scolymus).

BACKGROUND: The artichoke is a perennial horticultural plant that belongs to the Compositae family. OBJECTIVE: To present case studies of 2 vegetable warehouse workers who developed occupational rhinitis and bronchial asthma by sensitization to artichoke. METHODS: Skin prick tests with common inhalants and foods were performed. Specific IgE to artichoke, Parietaria judaica pollen, and Olea europaea pollen extracts was measured by a specific IgE enzyme immunosorbent assay kit. Molecular mass of the allergens was studied by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting technique. Patients underwent a nasal challenge test, and one patient provided peak expiratory flow rate (PEFR) measurements in her workplace. RESULTS: In both patients, results of skin prick tests to artichoke were positive. Levels of specific IgE for artichoke were 0.68 kU/L in patient 1 and 2.14 kU/L in patient 2. The protein composition of the artichoke extract, studied by SDS-PAGE, showed that most bands ranged from 30 to 14 kDa. The IgE-binding bands with the serum samples of patient 1 showed apparent molecular masses of 56, 48, 38, 31, 27, 25, 16, and 15 kDa; however, the serum samples of patient 2 showed IgE bands of 21 and 19 kDa. Western blotting of artichoke extract showed a complete inhibition of IgE-binding bands when serum samples were preincubated with P. judaica pollen extract. Nasal challenge with artichoke extract triggered a peak nasal inspiratory flow decrease of 81% and 85% in patient 1 and patient 2, respectively. Finally, patient 1 recorded a PEFR decrease of up to 36% after exposure to artichoke in her workplace. CONCLUSIONS: SDS-PAGE immunoblotting inhibition performed for the artichoke extract showed a total disappearance of the specific IgE binding bands when serum samples were previously incubated with P. judaica pollen extract, thus establishing the existence of a serologic cross-reactivity between artichoke and P. judaica pollen.