鸡蛋过敏原分离、鉴定与纯化

目的 对鸡蛋中主要过敏原进行分离、鉴定与纯化.方法 分别提取鸡蛋的蛋清与蛋黄的蛋白,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离鸡蛋的蛋白质组份,采用免疫印迹(Western-blotting,wB)方法鉴定过敏原,通过离子交换层析对鸡蛋主要过敏原进行初步纯化.结果 WB检测显示,蛋黄与鸡蛋过敏患者的阳性混合血清没有反应;蛋清中有4条蛋白带起反应,分子量分别为83,71,38,13 kD,尤以13 kD的蛋白质最为明显,离子交换层析可初步纯化出13 kD的过敏原蛋白.结论 鸡蛋的主要过敏原为13 kD的蛋白质,为临床诊断和治疗鸡蛋过敏性疾病提供标准化的过敏原提供了基础依据.     

点带石斑鱼过敏原分离、鉴定与纯化

目的 对点带石斑鱼的主要过敏原进行分离、鉴定与纯化.方法 通过十二烷基硫酸钠-聚丙炳酰胺凝胶电泳(SDS-PAGE)分离点带石斑鱼的蛋白质组份,采用免疫印迹(western-blotting)方法鉴定过敏原,通过离子交换层析对主要过敏原进行初步纯化.结果 SDS-PAGE结果显示,点带石斑鱼粗提液含有13条蛋白带,含量高的有8条,分子量分别为43,34,28,25,22,17,15,9 kD;westem-blotting显示,对点带石斑鱼过敏患者的阳性混合血清能与其中4个蛋白条带起反应,分子量分别为34,28,24,22 kD.离子交换层析可初步纯化出22 kD的过敏原蛋白.结论初步分离得到分子量为22 kD的点带石斑鱼主要过敏原,为临床诊断和治疗鱼类过敏性疾病提供理论依据.     

腰果主要过敏原分离鉴定与纯化

目的 对腰果的主要过敏原进行分析、鉴定与纯化.方法 通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)电泳分离腰果的蛋白质组份,采用免疫印迹(Western-blotting)方法鉴定过敏原,通过离子交换层析对腰果主要过敏原进行初步纯化.结果 腰果粗提液SDS-PAGE显示有10条蛋白条带,Western-blotting显示对腰果过敏患者的阳性混合血清能与其中2个蛋白条带起反应,分子量分别为21kD和11kD.离子交换层析可初步纯化出21和11kD的过敏原蛋白.结论 对腰果过敏原成功进行分离和鉴定,并初步纯化出腰果的主要过敏原.     

枝孢芽枝菌主要过敏原的分析与免疫、质谱鉴定

目的分析并用免疫和质谱方法鉴定枝孢芽枝菌(Cladosporium cladosporioides)的主要过敏原。方法空气中暴皿获得枝孢芽枝菌菌种,经鉴定后大规模培养;用碳酸盐缓冲液提取枝孢芽枝菌蛋白,并用SDS-PAGE、Western-blotting方法鉴定出枝孢芽枝菌的主要过敏原。主要过敏原胶内酶切,再经基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析,所得质谱数据进入网站(http://www.matrixscience.com)Mascot检索比对。结果经过暴皿获得枝孢芽枝菌菌种,其蛋白粗提液SDS-PAGE结果显示有14个条带,含量丰富的有10个条带;Western-Blotting鉴定出18kD的蛋白阳性率达66%,为枝孢芽枝菌的主要过敏原;质谱显示枝孢芽枝菌主要过敏原与约18kD嗜冷杆菌(Psychrobacter arcticum)50S的核糖体蛋白的匹配分值最高。结论采用免疫、质谱法鉴定了枝孢芽枝菌的主要过敏原是约18kD的核糖体蛋白。     

人心肌肌钙蛋白T的提取和纯化及初步鉴定

目的 从人心肌组织中提取、纯化心肌肌钙蛋白T，并进行初步鉴定。方法 采用匀浆、高盐提取、柱层析的方法。从心肌组织中分离、纯化cTnT。以凝胶电泳、免疫印迹进行鉴定。结果 cTn粗品经柱层析纯化，其第Ⅱ峰为cTnT活性峰，分子量约为34kD。结论 本实验获得纯度较高的cTnT，为进一步实验研究奠定了基础。     

不同温度下鱼类食品过敏原免疫学特性分析

目的研究鱼类食品在生熟状态下的过敏原组分，分析其热稳定性过敏原。方法4种新鲜鱼类经不同温度处理后去鳞、内脏和鱼骨。在预冷丙酮中破碎，脱脂，Coca＇s液提取其生熟总蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，考马斯亮蓝显色分析其总蛋白组成，用鱼过敏患者血清对不同温度条件下的鲩鱼总蛋白进行免疫学特征分析。结果SDS-PAGE显示，生熟鱼总蛋白提取物中均含有多种蛋白，经高温处理后熟鱼总蛋白组分相对减步。免疫印迹试验结果表明，加工温度、时间对鱼类食品过敏原有显著影响。结论鱼类食品中禽有热稳定的过敏原。即使加工后仍具有致敏性。     

鲤鱼主要变应原的分离、鉴定与纯化

目的 对常见的食物过敏原鲤鱼进行特异性抗原成分的分离、鉴定和纯化，为临床诊断及治疗鲤鱼过敏症提供理论依据。方法 通过磷酸盐缓冲溶液进行提取、用十二烷基硫酸钠．聚丙烯胺凝胶电泳（SDS-PAGE）分离出多种蛋白组分并测定其分子量；采用蛋白印迹技术（Western-blotting）鉴定其变应原并通过DE52离子交换层析对鲤鱼变应原进行初步纯化，进一步经凝胶过滤层析对变应原混合组分进行纯化。结果 SDS-PAGE分离出14种蛋白组分，其中主带有9条，分子量分别为5，46，36，34，26，25，23，20，16kDa。Western—blitting结果显示，鲤鱼的主要变应原及次要变应原的分子量是42，36kDa的变应原组分。结论 鲤鱼主要特异性变应原为42和36kDa的蛋白质，为临床诊断和治疗鱼类过敏性疾病提供标准化的廊原尊定了基础．     

鳙鱼过敏原小清蛋白基因的克隆表达、纯化及免疫学鉴定

目的克隆、表达与纯化鳙鱼(Aristichthys nobilis)过敏原小清蛋白,并检测免疫学活性。方法提取鳙鱼的总RNA,设计简并引物,采用RT-PCR方法扩增目的基因,克隆入T载体中进行测序和分析。将目的基因克隆到pET-28a(+)表达载体中于E.coli BL21(DE3)表达。通过Ni2+亲和层析,对重组蛋白进行纯化,采用免疫印迹(Western-blotting)方法检测其IgE结合活性。结果获得了鳙鱼过敏原小清蛋白基因,该基因被GenBank收录,登陆号FJ013047。基因开放阅读框共330个碱基(包括终止密码子),编码109个氨基酸,相对分子质量为11 537。Western-blotting结果表明,纯化后的重组蛋白能与过敏性患者血清中的特异性IgE结合。结论成功克隆、表达、纯化鳙鱼过敏原小清蛋白,此重组蛋白具有良好的免疫原性。     

腰果主要过敏原Anao2基因克隆及原核表达

目的 克隆腰果主要过敏原Anao2基因,并利用pMAL-c表达载体表达该蛋白.方法 提取腰果总RNA,设计特异性引物,反转录-聚合酶链反应(RT-PCR)克隆腰果Anao2基因,将其反转录基因连入pMD18Tsimple vector,提取质粒、双酶切、鉴定并测序;将测序正确的片段连入原核表达载体pMAL-c,将重组质粒转入BL21宿主表达菌中,丙基-β-D-硫代吡喃半乳糖苷诱导表达目的蛋白Anao2.结果 测序结果表明克隆腰果Anao2基因片段全长为1 332 bp,编码443个氨基酸,与GenBank中蛋白序列完全相同;对获得的重组蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定,目的蛋白大小与理论值相符.结论 成功克隆并表达了腰果过敏原Anao2.     

紫红笛鲷过敏原小清蛋白基因克隆及序列分析

目的 克隆紫红笛鲷(Lutianus argentimaculatus)过敏原小清蛋白(parualbumin)基因.方法 采用生物信息学方法对多种鱼的小清蛋白基因进行序列性比对,根据序列保守区域设计并合成简并性引物,通过实时(RT)-PCR和3'cDNA末端快速扩增RACE技术克隆紫红笛鲷小清蛋白的全长基因,并进行序列分析.结果 获得紫红笛鲷全长608 bp的新基因,开放阅读框为330个碱基,编码109个氨基酸,经分析该蛋白分子量约为11.5kD,等电点为4.35.序列分析结果显示所克隆的基因与其他鱼类过敏原小清蛋白基因有很高的同源性.结果 成功克隆出紫红笛鲷过敏原小清蛋白基因(登录号为:EF591789).     

刀额新对虾变应原的分离、鉴定与纯化

目的通过对我国常见的刀额新对虾变应原蛋白进行分离、鉴定与纯化，以提供虾特异性变应原用于食物变态反应疾病的诊断和治疗。方法通过十二烷基硫酸钠一聚丙烯酰胺凝胶电泳（SDS—PAGE）分离刀额新对虾的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刀额新对虾变应原进行初步纯化，通过疏水层析和凝胶过滤层析进一步对变应原纯化。结果刀额新对虾有17条蛋白带，其中主要条带有10条，68和36kD为可与虾过敏性病人血清IgE结合的特异性变应原；通过各种层析方法纯化出刀额新对虾68kD变应原，纯度为96％。结论对刀额新对虾变应原进行分离、鉴定和纯化，得到高纯度的68kD刀额新对虾变应原。为临床虾变态反应疾病的诊断和治疗奠定基础。     

舟山养殖大黄鱼烂尾病中哈氏弧菌的分离鉴定及药敏试验

目的：分离危害舟山海水网箱养殖大黄鱼烂尾病的病原。方法：从患病大黄鱼中分离的病原菌参照文献。进行分类鉴定、人工感染和药敏试验。结果：从患病大黄鱼中分离到2个菌株，根据形态及生理生化特征，其中1株S030901菌株属于哈氏弧菌(Vibrio Harveyi)，经人工感染试验证实能导致试验鱼100％死亡；另1株S030902菌株属于溶藻弧菌(V.alginolyticus)，对试验鱼几乎无毒性。药敏试验结果显示，S030901菌株对诺氟沙星等17种药物高度敏感，对头孢拉定等7种药物中度敏感，对青霉素G等16种药物不敏感。结论：哈氏弧菌是舟山海水网箱养殖大黄鱼烂尾病的病原菌，诺氟沙星、氟苯尼考等药物可作为治疗药物。     

海水网箱养殖大黄鱼烂尾病病原研究

[目的]研究危害舟山海水网箱养殖大黄鱼烂尾病的病原,为制订有效的防治措施提供技术依据。[方法]取深水养殖网箱之患病鱼分离病原菌,进行人工感染试验和药敏试验。[结果]从病鱼尾鳍和肌肉病灶分离到2个菌株,人工感染试验证明S030901菌株能导致试验鱼100%死亡,S030902菌株对试验鱼几乎无毒性。根据菌株的形态和生理生化特性实验结果,S030901菌株为哈氏弧菌(Vibrio.Harveyi),S030902菌株为溶藻弧菌(Vibrio alginolyticus)。药敏试验结果,S030901对诺氟沙星等17种药物高度敏感,对头孢拉定等7种药物中度敏感,对青霉素G等16种药物不敏感。[结论]哈氏弧菌是舟山海水网箱养殖大黄鱼烂尾病的病原菌。     

Soybean allergens and hypoallergenic soybean products

About 15 soybean proteins were shown to be recognized by sera of soybean-sensitive patients with atopic dermatitis. Three of them were identified as major allergens and designated as Gly m Bd 60K, Gly m Bd 30K, and Gly m Bd 28K, respectively. Gly m Bd 60K is an alpha subunit of beta-conglycinin well known as a major soybean storage protein. Gly m Bd 30K is also known as a soybean oil-body-associated glycoprotein with a molecular weight of 34,000, which is homologous to Der p (or f) 1, a major allergen of house dust mite, classified under the papain super family. Gly m Bd 28K is a vicilin-like glycoprotein with a molecular weight of 26,000, a minor component fractionated into 7S globulin fraction. The reduction of allergenicity of soybean and soybean products has been developed with respect to the above-mentioned major three allergens as the targets by the use of the combined techniques of a chemical breeding, a physico-chemical treatment, and an enzymatic digestion. Among the three major allergens, the alpha subunit of beta-conglycinin and Gly m Bd 28K were eliminated from soybean seeds by the development of a mutant line, Tohoku 124, introduced by a chemical breeding technique. The strongest allergen, Gly m Bd 30K, was almost completely removed from defatted soymilk prepared from Tohoku 124 by a salting-out technique and a centrifugation under the limited pH and ionic strength and alternatively by an enzymatic digestion. By the application of these procedures, several hypoallergenic soybean products, such as cooked soybean grains, soybean curd (Tofu), and fermented soybean paste (Miso), soymilk, and a jelly-like soybean cake have been made to evaluate their usefulness by a challenge test for soybean-sensitive patients. It has been demonstrated by a preliminary trial that about 80% of the soybean-sensitive patients could ingest these hypoallegenic products without any adverse reactions.     

Allergy to house dust mites and asthma

House dust mites have been shown to be important sources of indoor allergens associated with asthma and other allergic conditions. Asthma is a chronic respiratory disease that affects millions of people worldwide, and numerous scientific studies have shown that the prevalence of asthma is increasing. The most common dust mite species around the world include Dermatophagoides pteronyssinus (Dp), Dermatophagoides farinae (Df), Euroglyphus maynei (Em) and Blomia tropicalis (Bt). Over the past three decades, many important allergens from these species have been identified and characterized at the molecular level. The biological function of several house dust mite allergens has been elucidated, with many of them showing enzymatic activity. However, Bt allergens remain the least studied, even though this mite is very common in tropical and subtropical regions of the world, including Puerto Rico. Therefore, it is very important to include Bt in diagnostic and therapeutic strategies for house dust mite induced allergy and asthma, particularly in areas where Bt exposure and sensitization is high. Recombinant DNA technology, as well as other molecular biology and immunological techniques, have played a fundamental role in advances towards a better understanding of the biology of house dust mites and their role in allergic diseases. This kind of study also contributes to the understanding of the complex immunologic mechanisms involved in allergic reactions. The development of effective diagnostic and therapeutic approaches depends on the continuity of research of house dust mite allergens. The objectives of this review are to describe the most important aspects of house dust mite allergy and to acquaint the scientific community with the latest findings pertaining to house dust mite allergens, particularly those derived from Bt.     

Effects of dietary conjugated linoleic acids on the growth and quality of large yellow croaker fish Pseudosciaena crocea (Richardson) in cages

The objective of this study was to investigate the effect of conjugated linoleic acids (CLA) in artificial feeds on the growth and quality of large yellow croaker (Pseudosciaena crocea) raised in cages. Two diet formulas with dietary CLA were developed, and a diet without of CLA served as a control. The effects of CLA (2% and 4%) on average daily gain per tail (DGT), feed conversion ratio (FCR), muscle lipid, and lean body mass (LBM) for a 60-day period were evaluated. The results from two independent experiments revealed that diets with 4% CLA significantly improved DGT and FCR (p<0.05). Muscle lipid significantly reduced as increase of CLA content in diets (p<0.01), correspondingly, the LBM significantly increased (p<0.05). This study demonstrates that CLA supplemental diet could be used to improve growth and quality of large yellow croaker raised in cages.     

Paraneoplastic pemphigus

Paraneoplastic pemphigus is an autoimmune bullous skin disease induced by underlying malignant or benign neoplasias. The diagnostic and immunological criteria of the disease were characterized by Anhalt et al. in 1990. Clinical symptoms are variable, consisting of polymorphous blistering skin eruption and severe, painful mucocutaneous ulcerations. In a subset of patients, only papular lesions develop, resembling lichen planus, or graft-versus-host disease; in some cases blisters may develop later. Severe dyspnea, progressive respiratory failure with clinical features of bronchiolitis obliterans is a rather frequent and severe complication. The diagnosis can be established with direct and indirect immunofluorescent studies and immunoblot analysis. The autoantigens identified to date include cytoplasmic proteins of the plakin gene family: envoplakin (210 kD), periplakin (190 kD), plectin (approximately 500 kD), desmoplakin I (250 kD), desmoplakin II (210 kD) and bullous pemphigoid antigen 1 (230 kD). The desmosomal cadherins: desmogleins 1 and 3, and desmocollins 2 and 3, as well as bullous pemphigoid antigen 2 (180 kD) and an undetermined 170-kD transmembranous antigen are also target autoantigens in the disease. The mortality rate is more than 90 percent. Beside treatment of the underlying tumor, a combination of systemic steroids with immunomodulators, cytostatic drugs, plasmapheresis, plasma exchange, intravenous gammaglobulin, or anti-CD20 monoclonal antibody (rituximab) may be the most appropriate treatment.     

Hypersensitivity about environmental chemicals--mainly about food allergy

The hypersensitivity of environmental chemicals and natural products has been reviewed. Among environmental chemicals, small molecular weight molecules work as hapten and cause immediate-type and delayed-type hypersensitivity. Among natural products, relatively lower molecular weight protein or glycoprotein (MW 10,000-70,000 kDa) work as allergen and cause mainly immediate-type hypersensitivity. In recent years, amino acid sequence of important natural allergens have been determined, and three-dimensional structure and IgE epitopes of some of these allergens have also been determined. The characteristics of both inhalation and food allergens have been summarized. As for food allergens, the stability of these proteins in simulated gastric fluid(SGF) was one of the most important characteristics. In the last parts, the approach to the assessment of allergenic potential of genetically modified foods has been summarized.