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。     

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.     

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。     

斑节对虾过敏原的分离、鉴定与纯化

目的 对斑节对虾的主要过敏原进行分析、鉴定与纯化.方法 通过SDS-PAGE电泳分离斑节对虾的蛋白质组份,采用免疫印迹(Western-blotting)方法鉴定过敏原,通过离子交换层析对斑节对虾主要过敏原进行初步纯化.结果 斑节对虾粗提液SDS-PAGE显示其主要蛋白条带主要有7条,Western-blotting显示对斑节对虾过敏患者的阳性混合血清能与7个蛋白条带起反应,相对分子质量分别为71 000、43 000、34 000、23 000、21 000、20 000和19 000,离子交换层析可初步纯化出相对分子质量为34000和21 000的过敏原蛋白.结论本实验对斑节对虾过敏原进行了分离和鉴定,并初步纯化出斑节对虾的主要过敏原.     

方斑东风螺过敏原的分离、鉴定与纯化

目的 对方斑东风螺的主要过敏原进行分析、鉴定与纯化.方法 通过SDS-PAGE电泳分离方斑东风螺的蛋白质组份,采用免疫印迹(Western-blotting)方法 鉴定过敏原,通过离子交换层析对方斑东风螺主要过敏原进行初步纯化.结果 方斑东风螺粗提液SDS-PAGE显示其主要蛋白条带主要有7条,Western-blotting显示过敏患者的阳性混合血清能与其中3个蛋白条带起反应,相对分子质量分别是56 000、28 000和22 000.离子交换层析可初步纯化出28 000和22 000的过敏原蛋白.结论 本实验对方斑东风螺过敏原进行了分离和鉴定,并初步纯化出方斑东风螺的主要过敏原.     

艾蒿花粉主要变应原的分离、纯化与鉴定

目的　对我国蒿属花粉中常见、重要的变应原艾蒿花粉进行分离、鉴定与纯化。方法采用不同的提取液得到艾蒿花粉粗浸液 ,经饱和 (NH4 ) 2 SO4 分级沉淀后用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 (Mr) ;采用Westernblot鉴定其主要及次要变应原 ;通过DEAE CelluloseDE 32离子交换层析 (ionexchangechromatography ,IEC)和SephadexG 75凝胶层析 (gelchromatography)对艾蒿花粉变应原进行纯化。结果　分离后得到 2 0多种蛋白质组分 ,其中Mr 为 5 8× 1 0 3、38× 1 0 3、2 5× 1 0 3、2 0× 1 0 3、1 6× 1 0 3等 5个条带蛋白含量最丰富 ;分离到的蛋白质组分中有 9种蛋白能与确诊的蒿属花粉过敏患者血清中蒿属花粉特异性IgE结合 ,其中Mr 为 6 2× 1 0 3、4 3× 1 0 3、38× 1 0 3的蛋白条带的结合率最高 ;经纯化后仅得到Mr 为 6 2× 1 0 3的主要变应原。结论　艾蒿花粉的主要变应原Mr 分别为 6 2× 1 0 3、4 3× 1 0 3和 38× 1 0 3,层析技术可以对Mr 为6 2× 1 0 3的主要变应原成分进行纯化。     

重阳木花粉相关变应原profilin基因的克隆、表达及免疫学鉴定

克隆、表达和纯化重阳木花粉相关变应原profilin基因,并对其免疫学活性进行鉴定。采用RT-PCR和3-’RACE技术获得整个profilin基因的开放阅读框,将其与PET28a载体连接并转化大肠杆菌E.cdiBL21(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。克隆获得了profilin的全长基因,开放阅读框为396个碱基(包括终止密码子),编码131个氨基酸。成功地构建了原核表达载体,并在大肠杆菌中大量地表达了profi-lin,纯化后的重组蛋白进行免疫印迹,结果显示重阳木花粉过敏患者血清对重组profilin反应呈阳性。     

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。     

BSA-ELISA初步检测短穗鱼尾葵花粉特异性IgE

目的对短穗鱼尾葵花粉粗浸液的主要变应原进行分析、鉴定。方法通过SDS-PAGE分析短穗鱼尾葵花粉蛋白质组份,采用Western blotting鉴定主要变应原,以短穗鱼尾葵花粉粗浸液包板,摸索出其包被浓度、血清稀释度和酶结合物浓度,采用BSA-ELISA法对短穗鱼尾葵花粉过敏患者血清进行初步检测,并与皮肤挑刺试验比较。结果短穗鱼尾葵花粉粗浸液SDS-PAGE显示有30余条蛋白条带,其中主要蛋白条带有10条,Western blotting显示5例短穗鱼尾葵花粉过敏患者的混合血清能与其中3条蛋白条带起反应,分子量分别是26000、14000和12000Mr。BSA-ELISA检测短穗鱼尾葵花粉特异性IgE,最适粗浸液稀释度为1：100,血清稀释倍数为1：5,生物素化抗体为1：1000,辣根过氧化物酶标记的链霉亲和素（strepavidin-HRP）为1：1000。在此条件下BSA-ELISA与浸液皮试比较,检出结果与皮试阳性患者血清符合率为90%,与皮试阴性患者符合率为80%,与健康人对照检测符合率为100%。结论本实验对短穗鱼尾葵花粉主要变应原进行了分离和鉴定,BSA-ELISA法测定结果与皮肤挑刺试验初步比较符合率较好。     

法桐花粉主要过敏原基因Pla a1重组表达及鉴定

目的:表达、纯化和鉴定法国梧桐花粉主要变应原基因Platanus acerifolia pollen allergen1(Pla a1)。方法:首先根据文献查找并在GenBank获取法国梧桐花粉主要变应原基因序列Pla a1,利用DNAStar软件进行密码子优化;合成全基因;将Pla a1与载体pET-44a连接后转入大肠杆菌Rosetta中进行诱导并优化目的蛋白表达;利用亲和层析法纯化该外源表达蛋白;应用Western blot,利用法桐花粉过敏患者血清鉴定纯化后的目的蛋白的抗原性。结果:成功构建了pET44a-Pla a1阳性质粒;获得了法桐花粉主要变应原重组蛋白Pla a1;对该重组蛋白进行了亲和层析纯化;免疫印记法表明重组蛋白具有一定的抗原性。结论:首次利用密码子优化的方法获得融合Strep TagⅡ的法桐花粉过敏原重组蛋白Pla a1,为制备高纯度变应原、重组低致敏过敏原及变应原核酸疫苗奠定基础。     

Effect of various stabilizing agents on Imperata cylindrica grass pollen allergen extract

BACKGROUND: Allergen extracts are unstable, heat labile or susceptible to proteases. Stability of allergen extracts is important for proper diagnosis and therapy of allergic disorders. OBJECTIVE: The present study was undertaken to determine the preservation and stabilization conditions of Imperata cylindrica (Ic) grass pollen extract. METHODS: The Ic extract was kept with 0.1 mepsilon-aminocaproic acid (EACA), 0.75 m sucrose, 5% glycerol, 0.03% human serum albumin (HSA) or 0.4% phenol for different time periods. The extracts were stored for 3, 6 and 12 months each at 4 degrees C, 4 degrees C with daily exposure to room temperature (RT) for 1 h, and RT. The quality of extracts was analysed by SDS-PAGE, Western blot, ELISA, ELISA inhibition and skin test. RESULTS: Extracts kept with EACA and sucrose retained most of the protein bands followed by glycerol as determined by SDS-PAGE and Western blot during all storage periods and conditions in comparison with standard extracts. The extracts kept with HSA, phenol and without preservative (WP) showed protein degradation below 33 kDa after 3 months storage at all conditions. However, a 67-kDa allergen was stable in these extracts. EACA extract required 75 to 120 ng of protein for 50% inhibition in IgE binding under different conditions, whereas standard extract required 70 ng for the same. ELISA also demonstrated high allergenic reactivity of EACA extract. ID test on allergy patients with EACA extract demonstrated same allergenic potency as that of standard extract. CONCLUSION: EACA is the best preservative/stabilizing agent of Ic pollen extract, followed by sucrose and glycerol. Ic extract kept with phenol, HSA and without preservative showed degradation within 3 months. EACA preserved extract is equally potent as that of standard extract up to 1 year's storage.     

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。     

Podocarpus gracilior and Callitris verrucosa—newly identified allergens that crossreact with Cupressus sempervirens

Thirty-six symptomatic patients, with positive skin reactions to Cupressus sempervirens pollen extract were skin-tested with pollen extracts of Podocarpus gracilior and Callitris verrucosa, of these 17 (47%) had positive responses to P. gracilior, nine (25%) to C. verrucosa, and six (17%) to both. None of the non-atopic healthy controls had positive reactions to either of the extracts. Radioallergosorbent test (RAST)-inhibition studies were performed with pooled sera from three patients. Fifty per cent inhibition was obtained with 11 μg protein of C. sempervirens, 54μg of P. gracilior, and 71 μg of C. verrucosa; however, when pollen extract of Olea europaea, an unrelated allergen, was tested, 265μg protein were needed to obtain 50% inhibition. One-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of pollen extracts from the three species revealed that they had several very similar protein bands. Using Western blot analysis, several closely related IgE binding proteins were identified in the three species. It was concluded that the pollen grains of P. gracilior and of C. verrucosa are potentially allergenic. The presence of common allergenic proteins indicate partial crossreactivity with C. sempervirens.     

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.     

人免疫缺陷病毒Ⅱ型(HIV-2)gag蛋白基因在毕赤酵母中的克隆表达

目的：实现HIV-2HCOgag重组蛋白的分泌表达，为研究HIV—2HCO gag蛋白结构与功能及亚单位蛋白疫苗研究打下基础。方法：将HIV-2HCOgag蛋白基因片段，与分泌型毕赤酵母表达载体pPIC9重组，构建了相应的重组表达质粒pPIC9-gag，转化GS115酵母菌，经MD／MM表型筛选、PCR扩增筛选阳性克隆，以甲醇诱导表达后进行SDS-PAGE分析及Wgtem blot证实。结果：获得了HIV—2HCOgag重组蛋白在巴斯德毕赤酵母系统中的分泌表达，表达产物可被HIV—2抗血清识别，分子量约为57kD。结论：pPIC9—gag重组质粒在甲醇营养型酵母菌中可经甲醇诱导产生HIV—2HCOgag蛋白，该蛋白具有强免疫学活性。     

Characterization of antigens and allergens of date palm (Pheonix dactylifera) pollen

Antigenic and allergenic components of date palm (Phoenix dactylifera) pollen were investigated to observe their effects on the skin test reactivity, lymphocyte blastogenesis and cytokine production in atopic and healthy individuals. Date pollen extracts were fractionated using SDS-PAGE and Sephacryl S-200 gel filtration. Western blotting of SDS-PAGE separated components with antiserum raised against whole pollen extract in rabbits revealed at least 22 immunoreactive bands ranging in molecular weight between 12 and 94 kD. The immunogenicity of the pollen extract was further confirmed by strong positive reactions in ELISA and Ouchterlony's double diffusion tests. Immunoblot analyses revealed IgG and IgE reactive components (14-94 kD for IgG and 12-90 kD for IgE) in the skin test-positive patients' sera against whole pollen extract. Fifteen of 60 atopics reacted positively to either whole or some fractions of date pollen extract when skin tested. In response to whole or components of date pollen extract atopic patients showed differential peripheral blood lymphocyte (PBL) proliferative response and cytokine (IL-2, IL-4) production when compared with PBL of normal subjects. Our findings strongly suggest that date palm pollen should be considered a reaginic component and should be included in the battery of allergens for determining the allergic status of atopic patients, particularly in those parts of the world where the date palm is grown commercially.