椰子花粉过敏原基因的克隆表达、纯化及免疫学鉴定

目的:克隆并表达椰子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技术克隆椰子花粉中泛变应原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获得整个椰子花粉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:克隆获得了椰子花粉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.经分析,这个序列编码的蛋白为小分子量酸性蛋白,等电点为4.61,分子量约为14 kD.此序列已被GeneBank收录,登陆号为EF173598.重组椰子花粉profilin在大肠杆菌中高效的表达和纯化后,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椰子花粉profilin,为该花粉profilin用于椰子花粉过敏诊断和免疫治疗提供了理论依据.     

王棕花粉过敏原基因的克隆表达、纯化及免疫学鉴定

目的 克隆并表达王棕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技术克隆王棕花粉中泛变应原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获得整个王棕花粉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 克隆获得了王棕花粉profilin的全长基因,由675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.经分析,这个序列编码的蛋白为小分子质量酸性蛋白,等电点为4.86,相对分子质量(Mr)约为14.2×103.此序列已被GenBank收录,登录号为EF173599.重组王棕花粉profilin在大肠杆菌中高效表达,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 成功克隆和表达了王棕花粉profilin,为王棕过敏的诊断和免疫治疗奠定了基础.     

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。     

芒果果实中泛变应原profilin的克隆、表达及免疫学活性鉴定

目的　克隆并表达芒果果实中泛变应原肌动蛋白抑制蛋白(profilin)并了解其免疫学活性。方法利用RT-PCR结合:RACE技术克隆芒果果实中泛变应原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获得整个芒果profilin的开放阅读框,将其与pET-28a载体连接并转化大肠杆菌Escherichia coli 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。结果克隆获得了芒果profilin的2个型。每个型的cDNA都包括一个编码131个氨基酸的开放阅读框。序列分析结果显示所克隆到的基因与许多水果和花粉的泛变应原profilin基因有很高的同源性(>70%)。根据变应原的命名规则,将它们分别命名为Man i 3.01和Man i 3.02,并将这两个基因提交到GenBank数据库中,其登录号分别为DQ270547和DQ400579。重组芒果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芒果profilin,并证明与芒果过敏者血清有特异IgE应答。     

荔枝果实中profilin的表达纯化及其与桦树花粉Bet v 2的交叉反应性研究

目的 表达和纯化荔枝果实profilin,并对其免疫学活性及交叉反应性进行研究.方法采用RT-PCR获得整个荔枝profilin的开放阅读框,将其与PET28a载体连接并转化大肠杆菌Escherichia coli 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,并通过ELISA抑制实验对其与桦树花粉profilin的交叉反应性进行研究.结果成功的构建了原核表达载体,并在大肠杆菌中大量的表达了荔枝profilin,纯化后的重组蛋白进行免疫印迹,结果显示15个荔枝过敏患者中有5个存在重组pofilin的特异性IgE抗体.ELISA抑制实验发现它们之间存在很高的交叉反应性.结论 成功的表达了荔枝profilin,经纯化后具有很好的免疫学活性,为进一步的深入研究奠定了基础.     

欧蓍草花粉主要过敏原Parj1的重组表达及鉴定

目的:克隆、表达和纯化欧蓍草花粉主要过敏原Par j1。方法:根据Parj1.0102在GenBank中的序列号获得其核苷酸和氨基酸序列,确定开放阅读框,采用DNAstar软件优化密码子,合成全基因,并克隆到表达载体pET-44a中,转化表达宿主大肠杆菌Rosetta,优化蛋白表达条件并进行亲和层析纯化和Western blot鉴定。结果:PCR扩增及重组质粒测序结果表明成功地构建了pET-44a+/Par j1.0102原核表达质粒。对表达菌表达条件进行优化,最终确定在30℃,IPTG浓度为1.0 mmol/L,诱导4小时时蛋白表达量最高,重组蛋白经亲和层析纯化,SDS-PAGE分析纯化产物在23 kD处有明显的条带。Western blot表明重组蛋白具有与StrepII标签抗体结合活性。结论:国内首次获得融合StrepII标签的Par j1.0102重组蛋白,为欧蓍草花粉过敏诊断及特异性免疫治疗奠定基础。     

平榛主要过敏原Cor h 1的克隆表达、纯化及免疫学鉴定

目的 克隆、表达榛(平榛)主要变应原基因Cor h 1,检测其免疫学活性.方法 提取平榛的总RNA,设计带有酶切位点的特异性引物,采用RT-PCR方法 扩增目的基因,将其克隆入T载体中进行测序和分析.采用RT-PCR获得整个短的开放阅读框并将目的基因克隆到大肠杆菌表达载体pET28a中进行表达.通过Ni2+亲和层析,对重组蛋白进行纯化,采用免疫印迹(Western-blot)方法 检测其lgE结合活性.结果 克隆获得了平榛的主要过敏原Cor h 1的基因,该基因被GenBank收录,登陆号为EU195058.基因开放阅读框为486个碱基(包括终止密码子),编码161个氨基酸.该序列编码的蛋白等电点为6.16,相对分子质量约为17 600.结论 成功地克隆和表达了平榛主要变应原Car h 1,蛋白具有良好的免疫原性.     

平榛主要过敏原一个片段区基因的克隆表达及免疫鉴定

目的:克隆表达平榛(Corh)主要过敏原Corh1的一个片段区基因,并纯化表达的蛋白及检测其免疫学活性。方法:采用生物信息学方法选取Corh1的主要抗原表位区,设计带有酶切位点的特异性引物,采用RT-PCR方法扩增目的基因,将其导入pMD18-T载体中测序。将测序正确的质粒双酶切,并将获得的片段基因导入pET-32a中表达。通过Ni2+亲和层析柱纯化重组蛋白,采用Westernblot方法检测其IgE结合活性。结果:克隆并获得了Corh1的主要表位区基因,基因开放阅读框为243个碱基,编码81个氨基酸,蛋白相对分子质量(Mr)约为9000。表达的蛋白以可溶性为主,纯化出的蛋白有较好的免疫原性。结论:成功地克隆表达了Corh1的主要表位区基因,蛋白具有良好的免疫学活性。     

淡水小龙虾主要过敏原原肌球蛋白基因的克隆、表达及其免疫活性鉴定

目的:克隆、表达淡水小龙虾肌肉中主要过敏原原肌球蛋白(tropomyosin),并对其免疫活性进行鉴定。方法:根据甲壳类的泛过敏原tropomyosin基因的高度保守性设计简并引物,通过RT-PCR克隆出淡水小龙虾虾肉中过敏原tropomyosin的全长基因。将该基因与pET-28a载体连接并转化大肠杆菌Rosetta(DE3),经诱导异丙基-B-D-硫代乳糖苷(IPTG)诱导表达,Ni2+亲和层析柱纯化后,用Western blot检测该重组蛋白的免疫活性。结果:序列分析显示该基因包括一个编码284个氨基酸的开放阅读框,与已知虾、蟹、龙虾等的过敏原tropomyosin基因有较高同源性(>80%)。根据过敏原的命名规则,将其命名为Proc 1,并提交GenBank数据库,登录号为FJ769183。重组小龙虾tropomyosin在大肠杆菌中能高效的表达,Western blot检测结果显示该重组蛋白具有良好的免疫活性。结论:研究成功表达了具有免疫活性的淡水小龙虾原肌球蛋白,为虾过敏性疾病的诊断和治疗奠定了基础。     

豚草花粉泛过敏原同源基因克隆与序列分析

目的　克隆豚草花粉泛过敏原同源基因。方法　采用生物信息学分析方法对众多的花粉过敏原基因进行序列同源性比较 ,以序列保守区域为依据设计合成简并引物 ,在特殊的RT PCR条件下 ,结合RACE技术对豚草花粉中的过敏原全长基因进行克隆 ;通过Northern杂交及序列分析初步确定基因产物是否为花粉过敏原。结果　获得了 3个新的全长基因。序列分析显示 :所得过敏原同源基因与数十种不同种属来源的过敏原肌动蛋白结合蛋白 (profilin)具有较高的同源性 ,初步认定其为泛过敏原 ,并暂命名为Amba 8(t)。Northern杂交证实该基因在花粉中表达。结论　采用本研究方法成功地在豚草花粉中克隆到 3个泛过敏原基因 ,为豚草花粉重组过敏原的蛋白质表达及标准化奠定了物质基础     

Cloning of the minor allergen Api g 4 profilin from celery (Apium graveolens) and its cross-reactivity with birch pollen profilin Bet v 2

BACKGROUND: Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients. A subgroup of celery-allergic patients shows IgE-reactivity with this minor allergen. To investigate the IgE-binding potential and cross-reactivity of celery profilin at the molecular level, this study was aimed at the cloning and immunological characterization of this allergen. OBJECTIVES: Cloning, expression and purification of profilin from celery tuber to characterize its immunological properties and its cross-reactivity with birch pollen profilin. METHODS: Cloning of celery profilin was performed by polymerase chain reaction using degenerated primers and a 5'RACE method for the identification of the unknown 5'-end of the cDNA. Expression was carried out in Escherichia coli BL21 (DE3) using a modified vector pET-30a. The recombinant profilin was purified by affinity chromatography on poly L-proline coupled to sepharose. Immunological characterization was performed by immunoblotting, EAST and IgE-inhibition experiments. RESULTS: The coding region of the cDNA of celery profilin was identified as a 399-bp open reading frame, coding for a protein of 133 amino acids with a calculated molecular weight of 14.3 kDa. The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71-82%) recently described as allergens. Celery profilin was isolated as highly pure nonfusion protein. The IgE-reactivity of celery profilin was similar to that of natural protein. Seven of 17 celery-allergic patients tested presented specific IgE-antibodies to the recombinant protein tested by immunoblotting. Inhibition experiments showed high cross-reactivity of IgE with both profilins from celery and birch pollen. Moreover, the biological activity of recombinant celery profilin was demonstrated by a histamine release assay. CONCLUSIONS: Celery profilin is an important allergenic compound in celery and shows high homology to birch pollen profilin, Bet v 2. According to the revised IUIS allergen nomenclature, we suggest naming the celery profilin Api g 4. In addition to the cross-reacting major allergens Api g 1 and Bet v 1, birch pollinosis and associated allergies to celery can therefore additionally be explained by the cross-reactivity between homologous profilins. Moreover, recombinant Api g 4 may be used for target-specific diagnosis and structural analyses.     

IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes

BACKGROUND: Soybean proteins are constituents of a number of food products and represent a panel of potential allergens. Thus far, little is known about the molecular characteristics of soybean allergens. OBJECTIVE: The aim of this study was to identify the soybean profilin by PCR-based complementary (c)DNA cloning and to elucidate its allergenic characteristics. METHODS: Highly degenerate profilin-specific primers were used to identify, by means of PCR, 2 soybean profilin isoforms (GmPRO1 and GmPRO2) by using soybean cDNA as a target. One isoform (GmPRO1) with a length of 394 bp corresponding to 131 amino acid residues was subcloned and expressed in fusion with the maltose-binding protein. Moreover, 3 overlapping recombinant soybean profilin fragments comprising amino acid residues 1-65, 38-88, and 50-131 were also prepared as maltose-binding protein fusion proteins. IgE-binding reactivity of the recombinant proteins and the cross-reactivity of soybean profilin with birch profilin was studied by immunoblotting, enzyme-linked allergosorbent assays (EASTs), and competitive inhibition experiments by using serum samples from 13 soybean-sensitized subjects. RESULTS: Results of immunoblot analysis, EAST, and EAST-inhibition experiments indicate the presence of profilin in soybean extract. The recombinant soybean profilin (rGly m 3) was recognized by IgE in 9 (69%) of the 13 sera tested. Only the full-length rGly m 3 was able to bind with IgE antibodies, whereas the 3 soybean profilin fragments did not show significant binding reactivity, indicating that the IgE binding to rGly m 3 depends on the integrity of a conformational structure, which was not present in the overlapping profilin fragments. The rGly m 3 cross-reacted with birch pollen profilin (Bet v 2), and the IgE binding to Bet v 2 could be inhibited by rGly m 3. CONCLUSIONS: rGly m 3 represents a new soybean allergen with well-characterized primary sequence, and its IgE-binding reactivity is mediated by conformational epitopes.     

PCR-based cloning, isolation, and IgE-binding properties of recombinant latex profilin (rHev b 8)

BACKGROUND: Profilin (Hev b 8) in natural rubber latex (NRL) has been assumed to be an important allergen. Since latex profilin has a molecular mass similar to two other latex allergens (Hev b 1 and Hev b 6.03) in the 14-kDa range, it is difficult to obtain sufficient amounts of purified native profilin for investigations and diagnostics. The present study aimed to produce recombinant latex profilin (rHev b 8) and study its IgE-binding reactivity. METHODS: A profilin-specific cDNA encoding the latex profilin from Hevea brasiliensis leaves was synthesized and subcloned, and the rHev b 8 was overexpressed in fusion with the maltose-binding protein (MBP) in E. coli. The IgE-binding reactivity of rHev b 8 was studied by immunoblotting, immunoblot inhibition experiments, and the Pharmacia CAP method, with 25 sera from health-care workers with latex allergy and 17 sera from latex-sensitive spina bifida patients. RESULTS: rHev b 8 was found to have 131 amino acids and a sequence identity of 75% with birch profilin (Bet v 2). Analysis by the CAP system revealed the presence of rHev b 8-specific IgE antibodies in two out of 17 sera from spina bifida patients and in five out of 25 sera (20%) from health-care workers. Two subjects of the latter group with rHev b 8-specific IgE showed negative results in the skin prick tests with tree-pollen extracts and had no IgE to rBet v 2, indicating the presence of IgE-binding epitopes on the Hev b 8-molecule which do not cross-react with birch profilin. Immunoblot inhibition assays using MBP-rHev b 8 as inhibitor confirmed the presence of latex profilin in the NRL extract. IgE binding to the native latex profilin could be completely inhibited by the MBP-rHev b 8. CONCLUSIONS: Latex profilin represents a minor allergen in NRL and may have IgE-binding epitopes different from Bet v 2.     

Allergenic reactivity of the melon profilin Cuc m 2 and its identification as major allergen

BACKGROUND: Melon allergy is commonly associated with oral allergy syndrome (OAS) and with hypersensitivity to pollens and other plant foods. No melon allergen responsible for these clinical characteristics has yet been isolated, although profilin has been proposed as a potential target. OBJECTIVE: To isolate natural and recombinant melon profilin, to evaluate its in vivo and in vitro reactivity, and to analyse its behaviour in simulated gastric fluid (SGF) and heat treatments. METHODS: A pool or individual sera from 23 patients, and an additional group of 10 patients, all of them with melon allergy, were analysed by in vitro and in vivo tests, respectively. Natural melon profilin (nCuc m 2) and its recombinant counterpart (rCuc m 2) were isolated by poly-l-proline affinity chromatography, and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization analysis, DNA sequencing of cDNAs encoding rCuc m 2, and immunodetection with anti-profilin antibodies. In vitro analysis included IgE immunodetection, specific IgE determination, ELISA-inhibition assays, and histamine release (HR) tests. In vivo activity of nCuc m 2 was established by skin prick testing (SPT). The effect of SGF and heat treatment on rCuc m 2 was followed by immunodetection, ELISA inhibition, and HR assays. RESULTS: Both purified forms of melon profilin were recognized by rabbit anti-profilin antibodies and IgE of sera from allergic patients, and showed molecular sizes typical of the profilin family. nCuc m 2 had a blocked N-terminus, whereas rCuc m 2 rendered the expected N-terminal amino acid sequence, its full protein sequence being highly similar (98--71% identity) to those of profilins from plant foods and pollens. The natural allergen displayed a slightly higher IgE-binding capacity than its recombinant counterpart. Specific IgE to nCuc m 2 and rCuc m 2 was found in 100% and 78% of the 23 individual sera analysed, respectively. nCuc m 2 evoked positive SPT responses in all (10/10) patients tested, and rCuc m 2 induced HR in two out of three sera assayed. SGF treatment readily inactivated rCuc m 2, as shown by its loss of recognition by anti-profilin antibodies, lack of IgE binding, and inability to induce HR. In contrast, heat treatment did not affect the IgE-binding capacity of rCuc m 2. CONCLUSIONS: Profilin is highly prevalent in melon-allergic patients, and promptly inactivated by SGF, as expected for an allergen mainly linked to OAS.     

Identification of a 36‐kDa olive‐pollen allergen by in vitro and in vivo studies

BACKGROUND: Ole e 1 has been considered the major allergen of olive (Olea europaea) pollen. Some other relevant allergens (Ole e 2, 3, 4, and 6) have been recently described. This work aimed to study the IgE-binding frequency of a 36-kDa protein from O. europaea pollen in a large population of olive-allergic patients, its allergenic reactivity in vivo, and its presence in olive pollens of different origin, as well as in other relevant allergenic pollens. METHODS: Identification of IgE-binding components from O. europaea pollen extracts was elucidated by inhibition of SDS-PAGE immunoblotting using recombinant profilin (Ole e 2) and Ole e 1 molecules. The IgE-binding frequency of the 36-kDa protein was estimated by Western blot in a sample of 120 sera from olive-allergic patients. The cutaneous test with the 36-kDa protein was performed by intradermoreaction in allergic patients and control subjects. RESULTS: Exactly 83% of the sera from O. europaea-allergic patients recognized a protein with an apparent molecular weight of 36 kDa, under reducing conditions. It was detected by sera from monosensitized and polysensitized patients, showing a higher IgE frequency than the major allergen Ole e 1 (59%) and the minor profilin (Ole e 2) allergen (27%). Similar reactivity rates (79%) was found by intradermal test. Extracts from olive pollens collected in California presented a much higher amount (around 16-fold on average) of the 36-kDa protein than those from pollens of Spanish origin. The presence of similar allergens was detected only in closely related species (Syringa, Fraxinus, Ligustrum), and not in other common allergenic pollens. CONCLUSIONS: The 36-kDa protein constitutes a major allergen for olive-sensitized patients, but it is not equally represented in O. europaea pollens of different origins.     

Patient-tailored cloning of allergens by phage display: peanut (Arachis hypogaea) profilin, a food allergen derived from a rare mRNA.

A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10(11) cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-(L-proline)-Sepharose and yielded 30 mg l(-1) culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.     

Tomato profilin Lyc e 1: IgE cross-reactivity and allergenic potency

BACKGROUND: To date, very little data are available about the nature of tomato allergens. Immunoglobulin E (IgE) cross-reactive profilins have been suggested to account for allergic symptoms in patients suffering from tomato allergy. METHODS: The cDNA of tomato profilin was amplified by reversely transcribed polymerase chain reaction (RT-PCR) from total RNA extracted from ripe tomato fruit. The gene was cloned into the pET101D expression plasmid and the protein was produced in Escherichia coli BL21. Purification was performed via poly-l-proline (PLP) affinity chromatography. IgE reactivity of recombinant tomato profilin was investigated by immunoblot and enzyme-linked immunosorbent assay. IgE-inhibition studies were performed to analyse cross-reactivity with other profilins. To determine the allergenic activity of the recombinant protein, basophil histamine release assays using sera of patients with adverse reactions to tomato were performed. RESULTS: Profilin was identified as a new minor allergen in tomato fruits. The recombinant tomato profilin comprises 131 amino acids and high sequence identity to other allergenic food and pollen profilins. It was shown to be IgE-reactive with a prevalence of 22% (11/50) in tomato-allergic patients. In patients with tomato allergy and multiple sensitization to other foods and birch pollen, IgE directed against tomato profilin showed a strong cross-reactivity with profilins from plant food sources and birch pollen. The tomato profilin was able to induce mediator release from human basophils. CONCLUSION: The tomato profilin is a minor allergen in tomato fruit. Thus, it shows biological activity, as confirmed by in vitro histamine release assays with human basophils and thereby has the potential to account for clinical symptoms in tomato-allergic patients.     

IgE reactivity to profilin in pollen-sensitized subjects with adverse reactions to banana and pineapple

BACKGROUND: The so-called 'latex-fruit syndrome' is a well-documented phenomenon in cross-reactive allergies. By contrast, there is a lack of information about allergy to exotic fruits in patients with a predominant pollen sensitization. Since the ubiquitous protein profilin has been identified as an allergen in natural rubber latex as well as in pollen-related foods, the aim of this study was to investigate the role of profilin in allergy to certain exotic fruits. METHODS: Recombinant profilins from banana and pineapple were cloned by a PCR technique after isolation of total RNA using degenerated profilin-specific primers. The unknown 5' ends of copy DNA (cDNA) were identified by rapid amplification of 5'cDNA ends (5'-RACE) and expression in Escherichia coli BL21(DE3) cells. The recombinant profilins were purified by affinity chromatography using poly-(L)-proline as the solid phase. IgE-binding capabilities were characterized by means of immunoblot and Enzyme Allergosorbent Test (EAST). The cross-reactivity to birch pollen profilin and latex profilin was studied by EAST as well as by immunoblot inhibition experiments. RESULTS: Both banana and pineapple profilin were found to consist of 131 amino acid residues with high amino acid sequence identity to known allergenic pollen and food profilins (71-84%). IgE binding to the recombinant profilins was observed in 7/16 sera from subjects with suspected banana allergy (44%) and in 8/19 sera from subjects with suspected pineapple allergy (42%). Inhibition experiments indicated similar IgE reactivity of natural and recombinant allergens. In addition, high cross-reactivity to birch pollen profilin Bet v 2 and latex profilin Hev b 8 was demonstrated by immunoblot inhibition as well as EAST inhibition experiments. CONCLUSIONS: Since a high IgE-binding prevalence of about 40% was obtained in both banana and pineapple allergy, we conclude that profilin is an important mediator of IgE cross-reactivity between pollen and exotic fruits.