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.     

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。     

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。     

凡纳滨对虾次要过敏原肌质钙结合蛋白的质谱鉴定及免疫交叉反应

目的:质谱鉴定凡纳滨对虾相对分子质量(Mr)为21 000的次要过敏原组分肌质钙结合蛋白(SCP),分析它与其他虾、蟹等甲壳类过敏原的免疫交叉反应,以阐明SCP可作为甲壳类食物过敏原检测、检验及脱敏的标准过敏原物质。方法:运用MALDI-TOF/TOF-MS鉴定凡纳滨对虾Mr为21000的过敏原组分,利用软件BLAST、ClustalW分析甲壳类食物中该蛋白的氨基酸序列同源性,同时用纯化的21 000过敏原免疫小鼠制备其特异性多克隆抗体,通过该抗体与其他8种甲壳类食物蛋白粗提液的Western blot法结果分析该过敏原的免疫交叉反应。结果:质谱鉴定结果显示,凡纳滨对虾21 000过敏原为肌质钙结合蛋白;氨基酸序列同源性分析显示其与斑节对虾、中国对虾、克氏原螯虾、细趾小龙虾、褐虾的SCP序列一致性为81%～100%;Western blot结果显示,针对SCP的特异性多克隆抗体与凡纳滨对虾、刀额新对虾、斑节对虾、口虾蛄、罗氏沼虾、克氏原螯虾、远海梭子蟹、锈斑鲟、中华绒螯蟹粗提液在SCP对应的21 000左右处均有反应条带。结论:凡纳滨对虾Mr为21 000的次要过敏原为肌质钙结合蛋白,其氨基酸序列与多种甲壳类具有很高的同源性以及较强的免疫交叉反应。     

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。     

河虾主要过敏组分的分离、纯化、鉴定及其致敏性分析

目的:鉴定河虾中的过敏原组分,对主要过敏原组分进行纯化并分析其致敏性强弱。方法:用磷酸盐缓冲液(PBS)制备河虾蛋白粗提液,将其与11例虾过敏症患者血清IgE进行Westernblot,鉴定各种分子量的河虾过敏原组分;河虾蛋白粗提液用硫酸铵沉淀、G-50凝胶层析和阴离子交换层析等方法纯化其主要过敏原组分,再用虾过敏患者血清IgE进行Westernblot鉴定;并将纯化的相对分子质量(Mr)为21000、36000、800003种主要过敏原组分与虾过敏症患者血清IgE做间接ELISA,以分析其致敏性强弱。结果:West-ernblot结果显示,河虾蛋白粗提液出现9个阳性条带;其中3种主要过敏原组分21000、36000、80000与患者血清的反应率为36.4%,63.6%,45.5%;将这3种Mr的蛋白组分分别做间接ELISA,结果显示21000、36000、80000过敏原组分与11例虾过敏症患者的混合血清IgE结合的吸光值都显著高于河虾蛋白粗提液。结论:河虾中至少存在9个过敏原组分;21000、36000、8000等3种蛋白组分为河虾的主要过敏原组分,其中以36000过敏原组分致敏率和致敏性最强。进一步研究将探明21000、36000、80000等3种河虾过敏原组分的共同抗原表位,以期为明确食物过敏原检测、临床诊断和虾过敏原疫苗设计提供基础。     

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.     

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。     

Ara h2三聚体重组蛋白的制备及其低致敏原性鉴定

目的制备花生主要过敏原Ara h 2三聚体重组蛋白并检测其过敏原性。方法利用分子生物学的方法将3分子的Ara h 2依次串联起来,并将其整合到原核表达载体pET-32a(+),再转化到感受态Origami中;然后利用IPTG诱导其表达;通过Ni2+亲和层析纯化三聚体重组蛋白;Western-blotting和ELISA检测目的蛋白的过敏原性。结果测序结果表明Trimer成功整合到pET-32a(+)上。三聚体重组蛋白纯化后经SDS-PAGE鉴定,蛋白大小与理论值相符。Western-blotting和ELISA结果表明Trimer与重组的Ara h 2(r-Ara h 2)蛋白相比,结合花生过敏病人混合血清中IgE的能力有所降低。结论成功制备花生主要过敏原Ara h 2三聚体重组蛋白,初步的体外实验表明该重组蛋白具有低致敏原的潜能。     

福氏志贺菌毒力蛋白IpaC的表达、纯化及免疫活性鉴定

目的表达、纯化福氏志贺菌毒力蛋白IpaC并对其免疫活性进行鉴定。方法将含有ipaC基因的pET32a．ipaC表达质粒载体转入大肠杆菌BL21（kDE3）中表达，表达产物进行SDS—PAGE鉴定，并采用QIA expressionist^TM蛋白纯化系统纯化IpaC蛋白；用纯化的蛋白免疫动物获得抗血清，Western-blot检测抗原的免疫活性。结果诱导后的表达产物经SDS—PAGE发现有一相对分子量约为63000的条带，其含量约占总蛋白量的11％，对融合蛋白进行纯化纯度可达90％以上，抗His的抗体Western-blot分析，发现特异性区带出现在63000u处，证明该蛋白是所要表达的融合蛋白，制备的免疫血清可与Ipac发生特异免疫反应。结论pET32a-ipaC重组表达质粒转入大肠杆菌后，可稳定、高效地表达毒力蛋白IpaC，且纯化后的蛋白具有免疫活性。     

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。     

Development of a polyclonal antibody specific to VP19 envelope protein of white spot syndrome virus (WSSV) using a recombinant protein preparation

The VP19 gene encoding a structural envelope protein of white spot syndrome virus was cloned into an expression vector and introduced into E. coli. The objective was to produce a recombinant VP19 structural protein. After induction, the recombinant VP19 protein (rVP19) was produced, purified by SDS-PAGE and used for immunization of Swiss mice for polyclonal antibody production. The mouse anti rVP19 antiserum had specific immunoreactivity to the viral antigen in WSSV infected Penaeus monodon as verified by immunohistochemistry and Western blot. The production of monoclonal antibodies against this rVP19 may be useful in order to combine with anti-VP28 monoclonal antibodies for enhancing the sensitivity of various WSSV serological assays.     

Isolation and characterization of Tha p 1, a major allergen from the pine processionary caterpillar Thaumetopoea pityocampa

BACKGROUND: Pine processionary caterpillars induce dermatitis by a toxic-irritative mechanism. The existence of true allergic reactions to allergens from these caterpillars has been recently demonstrated by positive immediate skin prick tests and specific IgE determination by immunoblotting using crude larval extracts. The aim of this work was to purify allergens from the crude larval extract in order to characterize IgE-binding proteins from these caterpillars. METHODS: Allergens were separated by ethanol gradient fractionation and reversed phase HPLC. The N-terminal amino acid sequence of a selected allergen was obtained after SDS-PAGE and transfer. The clinical relevance of this allergen was measured using sera from patients allergic to caterpillar. RESULTS: An allergen with a molecular weight close to 15 kDa was purified. It was recognized by 9 out of 11 allergic patients (82%). Its N-terminal amino acid sequence had no homologies to any other protein already described in data bases. For this reason, no information about its biological function could be obtained. CONCLUSIONS: This 15-kDa IgE-binding protein is a major caterpillar allergen and shows no homologies to other insect allergens already described.     

N-terminal sequences of high molecular weight allergens from celery tuber

BACKGROUND: Celery tuber is an important source of food allergens. Low molecular weight celery allergens were identified as homologues of Bet v 1 and profilin. Little is known about the relevant allergens with molecular weights between 45 and 60 kDa, which cross-react with other plant food and pollen allergens. OBJECTIVE: The aim of this study was to isolate cross-reactive, high molecular weight allergens from celery and to characterize them by N-terminal sequencing. METHODS: High molecular weight allergens of celery were identified by immunoglobulin (Ig) E immunoblotting with patients' sera, and the IgE-binding patterns were compared with those of the monoclonal antibirch pollen antibody BIP3, as well as of a polyclonal rabbit anti-Art v 1 antiserum. Two independent methods, elution from preparative SDS-PAGE or anion exchange chromatography, were used to purify the IgE-binding celery proteins of interest. The isolated proteins were examined by N-terminal sequencing and IgE-immunoblots. RESULTS: Celery allergens with molecular masses of 55, 58 and 63 kDa, which were also recognized by the monoclonal BIP3 antibody and a polyclonal anti-Art v 1 antiserum, were isolated. The 63-kDa allergen was N-terminally blocked. The 55- and 58-kDa compounds yielded the same N-terminus, which showed no homology to known proteins in the databases. CONCLUSION: The combination of two independent protein separation techniques, immunoblotting and N-terminal sequencing, identified an N-terminus of two allergens in the 60-kDa molecular weight region. Our data will be helpful for the definite molecular characterization of these important cross-reactive molecules.     

Purification and IgE binding capacity of Der s 3, a major allergen from Dermatophagoides siboney

Background Sensitization to the house dust mite Dermatophagoides siboney has been demonstrated in asthmatic patients. Previously, Dermatophagoides siboney group 1 and group 2 allergens, named Der s 1 and Der s 2, respeetively, have been purified. Objectives The aim of this study was to purify and to study the IgE reactivity of a 30 kDa component, suspected to correspond to group 3 allergens. Methods The protein was purified by affinity chromatography using anti-Der f 3 monoclonal antibodies and semi-preparative SDS-PAGE. The IgE binding capacity of the purified fractions was tested with sera from 106 mite- sensitive asthmatic patients using a modified chemiluminiscent method. Results Affinity chromatography resulted in fractions containing the 30 kDa component which was further purified to homogeneity by SDS-PAGE. Seventythree per cent of the sera showed IgE reactivity to this protein, indicating that it is a major allergen. The protein also reacted with anti Der f 3 polyclonal antibodies and had tryptic activity. There were differences in the reaetivity to Der s 3 according to the age of the patients. Conclusion Based on the frequency of IgE reactions and the reactivity with antibodies directed to Der f 3, it is proposed to name this 30 kDa allergen from D. siboney, Der s 3.