鲤鱼小清蛋白的纯化及其过敏原性鉴定

目的：分离纯化鲤鱼小清蛋白(parvalbumin),并对其进行过敏原性鉴定,为建立鱼类过敏原检测技术奠定基础。方法：采用硫酸铵分级盐析和阴离子交换层析纯化鲤鱼小清蛋白,采用点杂交和特异性IgE 检测试剂盒筛选鱼类过敏者血清,应用聚丙烯酰胺凝胶电泳和免疫印迹技术分析确定纯化目标蛋白的性质。结果：硫酸铵分级盐析和阴离子交换层析纯化方法可以得到电泳纯单一目标蛋白;小鼠抗蛙小清蛋白单克隆抗体免疫印迹实验表明,所得纯化蛋白是小清蛋白。此外,免疫杂交结果显示,鱼类过敏者血清能与纯化的小清蛋白发生特异性结合,从而证实了鲤鱼小清蛋白的过敏原性。     

鲤鱼小清蛋白多克隆抗体的制备及免疫交叉反应的研究

小清蛋白是鱼类的主要过敏原,对小清蛋白的抗原性研究将有助于进一步深入理解淡水鱼过敏原在致敏过程中的免疫学意义。以鲤鱼为原材料,首先纯化出鲤鱼小清蛋白,再制备兔抗鲤鱼小清蛋白多克隆抗体,采用间接ELISA法测定抗体效价和抑制性ELISA法测定其特异性,然后采用免疫印迹法及抑制性ELISA法研究鲤鱼与青鱼、草鱼、鲢鱼之间的免疫交叉反应。结果表明,实验兔对鲤鱼类小清蛋白发生了高强度的免疫应答,应答多克隆抗体效价可高达720000,并且4种鱼类小清蛋白在12 ku处发生了强烈的免疫交叉反应。     

三文鱼过敏原小清蛋白分离纯化 及免疫结构鉴定

小清蛋白是鱼类的主要过敏原,三文鱼小清蛋白具有不同的亚型,分子量相近,序列基本相同,给分离纯化带来了不小的挑战。本试验采用硫酸铵盐析、Qxl-Sepharose离子交换结合凝胶过滤等方法定向纯化小清蛋白β1型,并应用免疫印迹法对纯化的小清蛋白进行过敏原性鉴定,结合激光辅助解析/飞行时间质谱进行结构鉴定。结果显示,硫酸铵盐析、离子交换层析结合凝胶过滤纯化方法可以得到纯度达90%以上的小清蛋白β1型,且蛋白具有很强的免疫反应活性。针对该蛋白的研究不仅有利于过敏原检测方法的建立,也可为低致敏性水产品的开发提供理论依据。     

离子交换层析法分离花生过敏原Ara h2的研究

为了制备出花生中重要过敏原Arah2,以生花生为材料,采用脱脂、离心、膜透析、离子交换层析等方法,纯化花生过敏原Arah2。结果显示,采用阴离子交换层析方法,制取的Arah2蛋白纯度达90%,得率为21.9%,该方法为过敏原Arah2的分离研究提供了可行的实验参数。     

牛奶过敏原的分离、鉴定与纯化

对牛奶过敏原进行分离、鉴定与纯化。通过SDS-PAGE电泳分离牛奶的蛋白质组份,采用免疫印迹(Western-blotting)方法鉴定过敏原,通过离子交换层析对牛奶过敏原进行初步纯化。结果表明,鲜牛奶粗提液SDS-PAGE显示蛋白条带有12条,奶粉粗提液SDS-PAGE显示出的蛋白条带与鲜牛奶的蛋白条带基本一致。鲜牛奶Western-Blotting显示14ku的阳性条带。离子交换层析可初步纯化出14ku的过敏原蛋白。本研究对牛奶过敏原进行了分离和鉴定,并初步纯化出牛奶的主要过敏原。     

芝麻过敏原的分离、鉴定与纯化

通过SDS-PAGE电泳分离芝麻的蛋白质组份,采用免疫印迹(Westem-blotting方法其鉴定过敏原,通过离子交换层析对芝麻主要过敏原进行初步纯化.结果表明白芝麻粗提液SDS-PAGE显示有14条蛋白条带.黑芝麻粗提掖SDS-PAGE显示有16条蛋白条带.Western-Blotting显示对芝麻过敏患者的混合阳性血清均能与黑芝麻、白芝麻11 ku蛋白反应,离子交换层析可初步纯化出白芝麻11 ku的过敏原蛋白.     

花生过敏原蛋白分离纯化方法研究进展

花生中已确定的过敏原蛋白包括Ara h 1～Ara h 11 11种。本文详细介绍花生中主要过敏原蛋白(Ara h 1、Ara h 2、Ara h 3/4、Ara h 6)以及非主要过敏原蛋白(Ara h 7～Ara h 11)的分离纯化方法研究进展。花生过敏原蛋白的分离纯化方法包括硫酸铵沉淀法、柱层析法、电泳法。其中硫酸铵沉淀法主要用于粗提纯化过程,而柱层析法则主要用于花生过敏原蛋白的精制,它包括离子交换层析、凝胶过滤层析、亲和层析、疏水相互作用层析、高效液相色谱。目前离子交换层析和凝胶过滤层析在花生过敏原蛋白分离纯化中应用最为广泛,而电泳法则仅见应用于Ara h 7及油质蛋白(Ara h 10、Ara h 11)的分离纯化。     

牛乳中主要过敏原的分离纯化研究进展

牛乳中含有3 种主要的过敏原蛋白：酪蛋白、β- 乳球蛋白和α- 乳白蛋白。本文详细叙述沉淀法、离子交换层析法、凝胶过滤层析法、羟基磷灰石层析法、疏水相互作用层析法、高效液相色谱法以及膜技术等分离纯化技术在牛乳主要过敏原分离纯化中的研究进展。其中,离子交换层析与凝胶层析已被广泛使用,而沉淀法一般作为粗提纯过的步骤。羟基磷灰石层析与疏水相互作用层析法也较为常见,既可单独分离过敏原,又可与其他方法结合来分离过敏原。另外高效液相色谱法与膜技术则是进一步纯化的后续工作,以提高过敏原的纯度。     

牛奶过敏原制备方法的研究和免疫活性鉴别

目的探索牛奶主要过敏原的制备工艺。方法采用等电点沉淀、凝胶层析和分子筛等技术纯化牛奶中主要过敏原组分;采用SDS-PAGE鉴定蛋白纯度,采用双抗原夹心-ELISA鉴定免疫活性。结果成功获得牛奶中的四种主要过敏原组分:酪蛋白、β乳球蛋白、α乳白蛋白和分子量较高的P1组分,并经过免疫实验证实这四种组分均能与过敏血清产生反应,而其中β乳球蛋白和P1组分反应性较高。结论本研究探索出一种简单、实用的牛奶主要过敏原制备工艺,并证实牛奶中的β乳球蛋白和高分子量蛋白质为主要过敏原。     

水产品过敏原及其消减方法研究进展

水产品在人类营养和健康中扮演重要角色。随着水产养殖业和国际贸易的发展,其市场和消费群体不断增多;由此水产品引发的过敏问题也逐渐增多,已成为世界性的重大食品安全问题之一。水产品的主要过敏原是小清蛋白(parvalbumin)和原肌球蛋白(tropomyosin,TM);另外,卵清蛋白、胶原蛋白(collagen)、精氨酸激酶(arginine kinase)、肌球蛋白轻链(myosin light chain)、肌钙结合蛋白(sarcoplasmic calcium binding pro-tein)等过敏原也扮演重要角色。基于此本文综述了水产品过敏原致敏机制、过敏原的种类、水产品致敏性的消减方法,并展望了水产品过敏原今后的研究方向和发展趋势。     

基于物种特异性PCR技术检测鱼糜中白鲢成分

目的建立一种基于物种特异性引物PCR测定混合鱼糜中白鲢鱼糜成分的快速检测方法。方法根据NCBI数据库中白鲢小清蛋白特异性较强的DNA序列位置设计引物进行PCR实验,通过测序得到了白鲢小清蛋白DNA的一段内含子序列,在此基础上设计了白鲢的特异性引物。提取样品DNA后进行PCR实验,产物经2%琼脂糖电泳分析进行引物特异性验证。结果在白鲢小清蛋白内含子位置设计的白鲢特异性引物,对巴沙鱼、铜盆鱼等8种鱼具有很强的物种特异性,可以实现对这9种鱼的混合鱼糜中白鲢成分的定性检测,且方法灵敏度为1%。结论本方法无需测序,能够快速、准确检测鱼糜中白鲢成分。     

Comparison of natural and recombinant forms of the major fish allergen parvalbumin from cod and carp

Allergic reaction following fish consumption can trigger life-threatening reactions in predisposed individuals. Parvalbumins from different species have been identified as the major fish allergens. There are two distinct phylogenetic lineages of parvalbumins, alpha and beta. Most allergic reactions are caused by beta-parvalbumins. We cloned and expressed cDNAs encoding cod (Gadus morhua) and carp (Cyprinus carpio) beta-parvalbumins and purified natural cod beta-parvalbumin. CD spectra of the purified proteins showed that their overall secondary structure contents were very similar. No differences in thermal stability were monitored in the calcium-bound or calcium-depleted form of natural cod parvalbumin. IgE reactivity was assessed using 26 sera of fish allergic patients from Spain, The Netherlands, and Greece in immunoblot and ELISA experiments. Twenty-five of the 26 patients with IgE reactivity to native and recombinant cod parvalbumin also reacted to the recombinant carp parvalbumin. IgE inhibition assays were performed using cod and carp extracts and purified recombinant parvalbumin of cod and carp. High crossreactivity among cod and carp parvalbumins was observed in immunoblots as well as in fluid phase assays. Natural and recombinant parvalbumins gave comparable results when performing various in vitro diagnostic assays.     

Purification of Parvalbumin from Carp: A Protocol that Avoids Heat-Treatment

ABSTRACT: Parvalbumin from carp, a major allergen, was purified to homogeneity using ion exchange chromatography and size exclusion chromatography (estimated purity >95% to 98% based on SDS-PAGE and native PAGE) with a yield of 318 mg, and a number of basic biochemical characteristics were determined. The identity was confirmed by peptide-mass fingerprinting, and IgE-binding was demonstrated. The UV/Vis absorbance spectra were explained using the previously published amino acid sequences. Far UV-CD spectroscopy was used to confirm the folding character of parvalbumin. We conclude that parvalbumin from carp can be purified on a comparatively large (hundreds of milligrams) scale using a purification protocol that does not include denaturing steps. The purified protein resembles biochemical characteristics as were earlier published for carp parvalbumin, that is, a molecular weight of approximately 12 kDa, amino acid sequence identity and a secondary structure containing α-helices and β-structures. The described method provides a yield sufficient to produce and characterize antibodies to construct immunochemical methods to detect parvalbumin in food, as well as for use as a standard calibrator for such assays. Practical Application: Parvalbumin is a major allergen from fish. Here, we have purified a comparatively large quantity from carp that can be used to develop antisera for use in an assay method to detect fish allergens.     

IgE reactivity to type I collagen and its subunits from tilapia (Tilapia zillii)

Acid-soluble collagen (ASC) was isolated from the skin of tilapia (Tilapia zillii) via acetic acid (HAc) extraction and NaCl precipitation. ASC from tilapia consists of α chains (α1 and α2), β chains and γ chains and is classified as type I collagen. A comparison of the properties of tilapia collagen and silver carp parvalbumin showed that tilapia collagen was less stable under heat treatment and more resistant to pepsin digestion. Both tilapia collagen and silver carp parvalbumin were degraded at pH 2.0 but stable at pH 3.0–11.0. Subunits α1 and α2 were further purified from tilapia collagen by carboxymethyl (CM) cellulose column chromatography with linear gradient elution and stepwise elution, respectively. Enzyme-linked immunosorbent assay (ELISA) and immunoblotting results demonstrated the specific IgE activity of different fish-allergenic patients’ sera towards the α1 and α2 chains of tilapia collagen. It can be inferred that tilapia collagen and its subunits are all potential allergens.     

红毛藻R-藻红蛋白的高效制备以及抗体标记与检测

采用硫酸铵盐析和DEAE-Sepharose阴离子交换柱层析相结合,从红毛藻(Bangia fusco-purpurea)中分离纯化到纯度系数(A565/A280)大于6.0的R-藻红蛋白。SDS-PAGE结果显示,其亚基分子量分别为19、21ku。用适宜摩尔浓度的异型双功能交联剂N-琥珀酰亚氨基-3-2-吡啶基二硫丙酸醇(SPDP)将纯化的R-藻红蛋白与羊抗小鼠IgG抗体交联,利用SDS-PAGE和荧光检测鉴定R-藻红蛋白与抗体的交联效果。结果显示:R-藻红蛋白能与羊抗小鼠IgG成功交联。分别采用Dotblot和Westernblot对R-藻红蛋白标记的抗体作为二次抗体,小鼠抗鲢鱼主要过敏原小清蛋白单克隆抗体为一次抗体;对鱼类小清蛋白进行免疫检测,结果显示:R-藻红蛋白标记的抗体能检测相关抗原的存在,且有良好的特异性。利用藻红蛋白荧光探针可缩短免疫杂交的检测时间,简化操作过程。     

Rapid Detection of Fish Major Allergen Parvalbumin by Surface Plasmon Resonance Biosensor

ABSTRACT: Seafood allergy is a common and major cause of food allergy in adults. In recent years, seafood allergy has become a serious problem with the increase of seafood consumption. To develop a rapid allergen detection method based on the affinity of antigen‐antibody interaction, fish major allergen, parvalbumin, was used for kinetic analysis by a surface plasmon resonance (SPR) biosensor. Anti‐parvalbumin murine monoclonal antibody (MAb) EG8 was immobilized onto a carboxymethyl dextran (CMD) surface. By the injection of various concentrations of purified carp parvalbumin (CPa), a standard curve and the affinity constants (K_D and k_*) for the MAb EG8‐CPa model system were determined. In addition, kinetic data were also obtained by the injection of serial dilutions of extracts from seafood products: sardine fish cake (tsumire) and dried skipjack tuna (katsuonut). Sardine tsumire and katsuonut contained 0.11 mg/kg and 0.39 mg/kg parvalbumins, respectively, where affinity constants K_D and k_* were almost similar among paralbumins from different sources. In the SPR system, the allergen can be detected only for 5 min according to the allergen‐MAb binding interaction. Consequently, by the use of a SPR biosensor, kinetic analysis based on the allergen specific MAb would be a rapid and powerful tool for allergen detection and quantification.     

Measuring parvalbumin levels in fish muscle tissue: relevance of muscle locations and storage conditions

Fish is an allergenic food capable of provoking severe anaphylactic reactions. Parvalbumin is the major allergen identified in fish and frog muscles. Antibodies against fish and frog parvalbumin have been used to quantify parvalbumin levels from fish. However, these antibodies react variably with parvalbumin from different fish species. Several factors might be responsible for this variation including instability of parvalbumin in fish muscle as a result of frozen storage and differential parvalbumin expression in muscles from various locations within the whole fish. We aimed to investigate whether these factors contribute to the previously observed variable immunoreactivity of the anti-parvalbumin antibodies. Results showed the detection of parvalbumin by these antibodies was unaffected by frozen storage of muscles for 112 days. However, the parvalbumin content decreased in fish muscles from anterior to posterior positions. This factor may partially explain for the inconsistent reactivity of anti-parvalbumin antibodies to different fish species.     

Parvalbumin in fish skin-derived gelatin: is there a risk for fish allergic consumers?

The major allergen parvalbumin was purified from cod muscle tissues, and polyclonal antibodies were raised towards it. The antibodies were tested for specificity and an enzyme-linked immunosorbent assay (ELISA) was developed using these antibodies. The ELISA was applied to measure parvalbumin in cod skin, the starting material for fish gelatin made from deep sea, wild fish. The ELISA was sufficiently sensitive (LLOQ?=?0.8?ng?ml(-1) in extracts, corresponding to 0.02?μg of parvalbumin per g of tissue), and did not cross-react with common food constituents. Fish gelatin, wine and beer, matrices for the potential use of this ELISA, did not cause disturbance of the assay performance. The data show that the parvalbumin content in cod muscle tissue is 6.25?mg?g(-1), while the skins contained considerably less, 0.4?mg?g(-1). Washing of the skins, a common industrial procedure during the manufacturing of fish gelatin, reduced the level of parvalbumin about 1000-fold to 0.5?μg?g(-1), or 0.5?ppm. From 95 commercial lots of fish gelatin it is shown that 73 are below 0.02?μg?g(-1) parvalbumin. From the other 22 lots, the one with the highest concentration contained 0.15?μg?g(-1) of parvalbumin. These levels are generally assumed to be safe for fish-allergic individuals.     

Parvalbumin in fish skin-derived gelatin: is there a risk for fish allergic consumers?

The major allergen parvalbumin was purified from cod muscle tissues, and polyclonal antibodies were raised towards it. The antibodies were tested for specificity and an enzyme-linked immunosorbent assay (ELISA) was developed using these antibodies. The ELISA was applied to measure parvalbumin in cod skin, the starting material for fish gelatin made from deep sea, wild fish. The ELISA was sufficiently sensitive (LLOQ?=?0.8?ng?ml^?1 in extracts, corresponding to 0.02?µg of parvalbumin per g of tissue), and did not cross-react with common food constituents. Fish gelatin, wine and beer, matrices for the potential use of this ELISA, did not cause disturbance of the assay performance. The data show that the parvalbumin content in cod muscle tissue is 6.25?mg?g^?1, while the skins contained considerably less, 0.4?mg?g^?1. Washing of the skins, a common industrial procedure during the manufacturing of fish gelatin, reduced the level of parvalbumin about 1000-fold to 0.5?µg?g^?1, or 0.5?ppm. From 95 commercial lots of fish gelatin it is shown that 73 are below 0.02?µg?g^?1 parvalbumin. From the other 22 lots, the one with the highest concentration contained 0.15?µg?g^?1 of parvalbumin. These levels are generally assumed to be safe for fish-allergic individuals.