热带剥爪螨变应原Blot 5的克隆表达、纯化及免疫学鉴定

目的:克隆热带剥爪螨主要变应原Blot 5基因,表达纯化该蛋白,并检测其免疫活性。方法:提取热带剥爪螨总RNA,采用RT-PCR的方法扩增Blot 5编码基因,将其连入原核表达载体pET-19b,将重组质粒转化入大肠杆菌BL21 Star(DE3)pLysS,IPTG诱导表达后,通过Ni2+亲和层析纯化重组变应原Blot 5。用Dot blot、Western blot和ELISA等方法检测重组变应原Blot 5免疫活性。结果:Dot blot和Western blot结果表明重组变应原Blot 5和热带剥爪螨粗提液都能和Blot 5鼠单克隆抗体结合。重组变应原Blot 5检测69份热带剥爪螨过敏患者血清和21份户尘螨过敏患者血清中的特异性IgE,阳性率分别为29.0%和33.3%。结论:表达和纯化了具有与天然蛋白相似的免疫活性的重组热带剥爪螨变应原Blot 5,可用于热带剥爪螨变应原的标准化,为标准化抗原的临床特异性诊断与治疗奠定基础。     

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。     

青蒿花粉变应原Art a1基因的克隆、表达及特性鉴定

目的克隆、表达和鉴定青蒿花粉变应原Arta1。方法在成功构建青蒿花粉cDNA文库的基础上,用蒿属花粉过敏患者的阳性混合血清进行免疫学筛选,所获阳性克隆亚克隆入pET24a(+),经IPTG诱导表达后,通过Ni2+亲和层析柱对重组变应原进行纯化,并采用Westernblot和ELISA检测其IgE结合活性。结果选用阳性血清从青蒿花粉cDNA文库中筛选到1个阳性克隆,经序列测定,该基因与GenBank中已知基因无明显同源性,含有长度为609bp的开放阅读框,编码203个氨基酸,命名为Arta1;该重组变应原在大肠杆菌中高效表达为相对分子质量(Mr)为22.7×103蛋白,进一步在Ni2+亲和层析柱得到高度纯化;免疫学分析表明重组变应原有良好的IgE结合活性。结论本研究克隆和鉴定了一个青蒿花粉主要变应原Arta1(登录号为:CK700713),为花粉过敏性疾病的诊断和免疫治疗及进一步的实验研究奠定基础。     

重组屋尘螨变应原突变体的免疫原性与过敏原性研究

目的 构建屋尘螨Der p2变应原突变体的原核表达质粒,并在大肠杆菌中表达、纯化,以观察屋尘螨Der p2变应原突变体的免疫原性以及抗原性.方法 基因敲除屋尘螨Der p2变应原的部分氨基酸残基序列,将编码基因定向克隆到pET32a原核表达质粒中,转入BL21表达菌中并提纯;将18只Balb/c小鼠随机分为Der p2变应原突变体组、重组Der p2组和对照组.采用酶联免疫吸附试验(ELISA)测定各组小鼠血清中IgE、IgG1与IgG2a抗体变化,用免疫斑点试验测定Der p2突变体的IgE结合能力.结果 成功构建了Der p2变应原突变体的原核表达质粒,表达并纯化了Der p2变应原突变体.腹腔免疫后0d,各组IgE、IgG1与IgG2a抗体表达无明显变化(P=0.067、P=0.052、P=0.078);免疫后第7d,重组Der p2变应原组诱导IgE抗体的产生量高于Der p2变应原突变体组(P =0.002),重组Der p2变应原组诱导的IgG2a抗体高于Der p2变应原突变体组(P=0.0056);腹腔免疫后第14 d,重组Der p2变应原组IgE抗体的产生量高于Der p2变应原突变体组(P=0.001),Der p2变应原突变体组IgG2a低于重组Der p2变应原组(P=0.0034).Der p2变应原突变体与屋尘螨过敏患者的阳性血清结合明显较低.结论 Der p2变应原突变体能诱导小鼠产生IgG1抗体和IgG2a抗体,同时能明显减少IgE抗体的产生,并且Der p2变应原突变体具有较低的IgE结合能力.     

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.     

多主枝孢霉重组变应原Cla h8的制备及其免疫活性鉴定

目的:通过基因工程手段,获得重组多主枝孢霉变应原蛋白Cla h8,有利于进行变应原的标准化,为标准化抗原的临床特异性诊断与治疗奠定基础。方法:从多主枝孢霉菌体中提取总RNA,采用RT-PCR的方法扩增Cla h8编码基因,将其连入pET-19b载体。转入大肠杆菌BL21 Star(DE3)pLysS,经诱导表达后,进行提纯复性,用Western blot和Dot-blot检测其免疫活性。结果:重组多主枝孢霉变应原Cla h8蛋白可以与多主枝孢霉过敏患者的血清中IgE和IgG抗体特异性结合,与天然蛋白具有相似的免疫活性。结论:制备并获得了具有生物学活性的可溶性重组多主枝孢霉变应原Cla h8蛋白,可用于多主枝孢霉变应原的标准化,克服天然提取物的非单一性及标准化难的障碍。     

支气管哮喘血清E-选择素与IgE的相关性研究

目的探讨支气管哮喘的发病机理。方法对23例哮喘患者及20例正常人用ELISA法测定其血清中可溶性粘附分子sE-selectin的含量,采用荧光免疫法,测定其血清总IgE(tIgE)、特异性IgE(sIgE)和嗜酸性粒细胞阳离子蛋白(ECP),并作相关性分析。结果哮喘组的sE-selectin和tIgE血清含量与对照组相比,差异有高度显著性(P<0.001)多因素相关性分析提示sE-selectin与tIgE存在直线正相关(r=0.664,P<0.002);蒿草花粉、屋尘螨、屋尘及複草花粉的血清sIgE水平较高,为引起哮喘发病的主要变应原;哮喘急性发作期的血清ECP水平明显高于缓解期(P<0.001)。结论哮喘患者血清可溶性粘附分子sE-selectin和IgE较正常人高,且存在直线正相关;我国北方地区引起哮喘的常年性变应原为屋尘螨和屋尘,而夏秋季的主要变应原是蒿草花粉和複草花粉;血清ECP含量在哮喘发作期较缓解期高,这可能与嗜酸性粒细胞处于活化状态有关。     

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。     

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.     

猫过敏原白蛋白的克隆表达、纯化和免疫学特性鉴定

目的 克隆、表达猫过敏原白蛋白(Fel d 2),并分析其过敏原活性.方法 提取猫肝脏总RNA,逆转录合成cDNA,采用适宜引物进行PCR扩增目的 基因,随后将其克隆到原核表达载体PET24a( ),在大肠杆菌中进行表达,镍亲和柱层析法提纯重组蛋白,Western blot检测其过敏原活性.结果 序列分析表明所克隆白蛋白的序列由1 752 bp组成,编码584个氨基酸,与Genebank上的基因序列(NM 001009961)同源性为99.9%.SDS-PAGE显示表达的重组白蛋白Mr65 000.免疫印迹表明重组白蛋白具有与猫过敏原过敏病人血清IgE结合活性.结论 表达的重组白蛋白具有与猫过敏患者血清IgE结合活性.     

Molecular cloning of a class IV chitinase allergen from Japanese cedar (Cryptomeria japonica) pollen and competitive inhibition of its immunoglobulin E-binding capacity by latex C-serum

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergic diseases in Japan. Only three C. japonica allergens, Cry j 1, Cry j 2, and CJP-6, have been characterized. The full IgE-binding spectrum of C. japonica pollen allergens demonstrates that many allergens remain to be identified. OBJECTIVE: The aim of this study was to characterize a novel allergen with a high frequency of IgE binding. METHODS: The cDNA coding for a high-frequency IgE-binding protein, designated CJP-4, was cloned from the total mRNA of C. japonica pollen. The corresponding native allergen was purified by affinity precipitation with colloidal chitin and gel chromatography. The IgE-binding ability of purified native CJP-4 was characterized by ELISA and ELISA inhibition. RESULTS: The CJP-4 cDNA encoded 281 amino acids with significant sequence homology to class IV chitinases. Purified native CJP-4, migrated as a homogeneous 34-kDa protein on SDS-PAGE, revealed endochitinase activity on native PAGE. The purified protein displayed the ability to bind IgE from all patients tested (31/31) in ELISA, whereas Cry j 1 bound to IgE at a 71% frequency (22/31). Pre-incubation with latex C-serum completely inhibited the reaction of pooled sera IgE from patients with C. japonica pollinosis and/or latex allergy to purified CJP-4. CONCLUSION: We identified CJP-4 as a novel and fourth C. japonica chitinase allergen with high IgE-binding frequency. The competitive IgE-binding profile between C. japonica chitinase and latex C-serum indicated that C. japonica chitinase should be an important pan-allergen in C. japonica pollen.     

Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. OBJECTIVE: The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. METHODS: C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. CONCLUSION: Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.     

尘螨变应原Der f 6 / pET32a( +) 重组质粒构建、表达、纯化及其产物血清 IgE 结合率

目的:获得尘螨变应原第6 组分Der f 6 原核表达产物并检测其与尘螨过敏性哮喘患儿血清抗体IgE 结合率。方法:酶切质粒pET28a(+)-Der f 6 获得目的基因Der f 6,将其与pET32a(+)载体连接成质粒pET32a (+)-Der f 6,转化BL21细菌后,用异丙基硫代半乳糖苷(IPTG)诱导表达,用Ni+离子亲和层析柱纯化表达产物,用十二烷基磺酸钠鄄聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫印迹实验(Western blot)和蛋白质串联质谱(MALDI-TOF/ TOF)鉴定纯化产物。以纯化获得的产物为包被抗原建立间接ELISA 法检测尘螨过敏性哮喘患儿血清抗体反应情况。结果:成功构建了原核表达质粒pET32a (+)-Der f6,将该质粒转化E.coli BL21 诱导表达,亲和层析纯化后,SDS-PAGE 显示获得目的蛋白,Western blot 验证其能够与载体的组氨酸标签结合,质谱鉴定其Der f 6 结构一致。以此产物为包被抗原建立间接ELISA 检测尘螨过敏性哮喘患儿血清,阳性率为41.3% (19/46)。结论:成功构建了原核表达质粒pET32a (+)-Der f 6,亲和纯化获得的目的蛋白具有良好的反应原性。     

家蚕过敏原Profilin 的表达、纯化、免疫学鉴定及其生物信息学分

目的：克隆表达家蚕(Bombyx mori) Profilin蛋白,鉴定其免疫原性并进行B细胞抗原表位预测和构建分子进化树。方法：从NCBI上获取家蚕Profilin蛋白的基因序列,合成该基因并构建到pet 28a表达载体上,重组质粒转化至E.coli BL21,IPTG诱导基因表达,通过亲和层析获取高纯度蛋白;用Western blot方法对重组蛋白进行过敏原性鉴定;通过DNAStar软件分析其潜在的B细胞抗原表位;应用MEGA5.05进行序列比对,并构建分子进化树。结果：成功表达出重组蛋白,且重组蛋白与家蚕过敏患者血清有一定的IgE结合。结论：成功克隆表达出具有免疫原性的Profilin蛋白,并成功预测出其B细胞抗原表位和构建出分子进化树。     

Protein sequence analysis of a novel 103-kDa Dermatophagoides pteronyssinus mite allergen and prevalence of serum immunoglobulin E reactivity to rDer p 11 in allergic adult patients

BACKGROUND: House dust mites are regarded as important indoor allergens. While the most studies mite allergens are low molecular weight (mw), a high mw Dermatophagoides farinae mite paramyosin (Der f 11) has recently been cloned. We have also cloned a novel high mw Dermatophagoides pteronyssinus (Dp) mite allergen, Der p 11. OBJECTIVE: The aim of this study was to isolate and express a cDNA gene coding for a Der p 11 allergen, to compare the sequence of Der p 11 with other antigens and to evaluate the presence of IgE reactivity to the recombinant protein (rDer p 11) in the sera of allergic adult patients. METHODS: The full-length Der p 11 gene was isolated by cDNA library screening, 5'-3' rapid amplification of cDNA ends and PCR. The cDNA gene was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The allergenicity of rDer p 11 was tested by human IgE immunodot or immunoblot assay in a large panel of 100 allergic patients with bronchial asthma, allergic rhinitis or eczema. RESULTS: Der p 11 is a 2965 bp cDNA gene with a 2625 bp open reading frame coding for a 875 amino acid protein. The deduced amino acid sequence of the Der p 11 showed significant homology with various invertebrate paramyosins. The prevalence of serum IgE reactivity to rDer p 11 on immunodot assay ranged from 41.7% to 66.7% in different allergic patient groups, whereas it was rare in non-atopic patients with urticaria (18.8%) and in normal individuals (8%). A high frequency (five out of eight) of MAST(Dp)- allergic serum samples had specific IgE-binding activity to rDer p 11 or its fragments on immunoblot assay, even though their IgE-binding activity to Dp extract was either weak or negative. CONCLUSION: The 103-kDa Der p 11 appears to be major Dp mite allergen with a high frequency of IgE reactivity in sera of patients allergic to mites.     

Isolation and characterization of native Cry j 3 from Japanese cedar (Cryptomeria japonica) pollen

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is the most prevalent allergy in Japan. Recently, the Japanese cedar pollen allergen Cry j 3 was cloned as a homologue of Jun a 3, which is a major allergen from mountain cedar (Juniperus ashei) pollen. However, native Cry j 3 has not been isolated and there are no reports on its allergenic activity. The aims of this study were to isolate native Cry j 3 and assess its immunoglobulin E (IgE)-binding capacity in patients with Japanese cedar pollinosis. METHODS: Native Cry j 3 was purified from Japanese cedar pollen by multidimensional chromatography. We assessed the IgE-binding capacity using sera from patients allergic to Japanese cedar pollen by immunoblot analysis and ELISA. Moreover, we assayed the capacity of Cry j 3 to induce histamine release from the patients' leukocytes. We cloned cDNA corresponding to purified Cry j 3 from a cDNA library of Japanese cedar pollen. RESULTS: We isolated native Cry j 3 as a 27-kDa protein. The IgE-binding frequency of Cry j 3 from the sera of patients allergic to Japanese cedar pollen was estimated as 27% (27/100) by ELISA. Cry j 3 induced the release of histamine from leukocytes. We cloned the cDNA and named it Cry j 3.8. Cry j 3.8 cDNA encoded 225 amino acids and had significant homology with thaumatin-like proteins. CONCLUSIONS: Cry j 3 is a causative allergen in Japanese cedar pollinosis and may play crucial roles in the cross-reactivity with oral allergy syndrome.     

Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae

BACKGROUND: The important dust mite allergens identified to date are of molecular weights ranging from 14 to 60 kDa. Our previous protein study indicated that the 98-kDa native paramyosin in Dermatophagoides farinae mite showed IgE reactivity with 82% of the mite-sensitive asthmatic patients suggesting that it is a novel major mite allergen. This study described the isolation and characterization of the cDNA clone encoding the 98-kDa mite allergen. METHODS: A Dermatophagoides farinae cDNA library was constructed in lambda ZAPII vector and the library was immunoscreened with a monoclonal antibody 642. The cDNA insert was sub-cloned into M13 sequencing vector for single-stranded sequencing. The whole cDNA insert was expressed in pGEX-2T Escherichia coli expression system as a fusion protein with GST. The allergenicity of the recombinant peptides was tested by skin tests and IgE immunoassay. The IgE and IgG immunoassays were performed with sera from 20 mite-allergic patients. RESULTS: The cDNA clone Df642 was 2134 bp long, coding for a polypeptide of 711 amino acid residues. Protein sequence analysis and alignment confirmed that the deduced polypeptide is a mite paramyosin which is truncated slightly at the N- and C-terminuses. In vivo skin tests and in vitro IgE-binding study showed that 62% (13/21) and 50% (10/20) of the mite-sensitive asthmatic patients reacted positively with the recombinant Dermatophagoides farinae paramyosin, respectively. CONCLUSION: The study indicated that 98-kDa mite paramyosin is an important allergen.     

The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50–60 kDa is comprised of two major allergens: Phl p 4 and Phl p 13

BACKGROUND: More than 70% of the patients allergic to grass pollen exhibit IgE-reactivity against the high molecular mass fraction between 50 and 60 kDa of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allergen, Phl p 13, has recently been purified and characterized at the cDNA level. OBJECTIVE: The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE-binding frequency and capacity. METHODS: Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects allergic to grass pollens were used to determine specific IgE-binding frequency to Phl p 4 and Phl p 13. IgE-binding of allergens was quantified by ELISA measurements. Pre-adsorption of sera with purified allergens and subsequent incubation of nitrocellulose-blotted timothy grass pollen extract was performed to determine whether or not Phl p 4 and Phl p 13 represent the whole high molecular mass allergen fraction. Proteolytic stability of both allergens was investigated by addition of protease Glu-C. RESULTS: More than 50% of 300 patients displayed IgE-binding with both allergens. Clear differences concerning the immunological properties of Phl p 4 and Phl p 13 were confirmed by individual IgE reactivities. Quantification of specific IgE for both allergens revealed comparable values. For complete inhibiton of IgE-binding in the high molecular mass range preincubation of sera with both allergens was necessary. Interestingly, inhibition of strong reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 kDa double band, but in addition a 'background smear'. Whilst undenatured Phl p 4 was resistent to proteolytic digestion with Glu-C, native Phl p 13 was degraded rapidly. CONCLUSION: Phl p 4 and Phl p 13 are immunologically different and must both be considered as major allergens. They are judged to be important candidates for potential recombinant therapeutics that may provide a basis for improved immunotherapy.     

Discontinuous IgE-binding epitopes contain multiple continuous epitope regions: results of an epitope mapping on recombinant Hol l 5, a major allergen from velvet grass pollen

The knowledge of IgE-binding epitopes on allergen molecules is important for better understanding allergen-antibody interactions and, thus, for developing new strategies for immunotherapy. Our purpose was to more precisely define the number and structure of IgE-binding epitopes of a paradigmatic major grass pollen allergen. We performed an IgE-binding epitope mapping of rHol l 5, a group V pollen allergen of velvet grass (Holcus lanatus), with overlapping fragments (length between 15 and 186 amino acids), which were expressed in E. coli as MBP fusion proteins. Using sera of 65 grass pollen allergic patients, the fragments were analysed by immunoblotting for IgE reactivity. Specificity of antibody binding was confirmed by competitive blot inhibition assays. At least four different continuous IgE-binding epitopes were identified on small fragments (about 30 amino acids), and at least five different discontinuous IgE-binding epitopes on larger fragments, which were destroyed by further fragmentation. The fragments were differentially recognized by individual patients' sera. By investigating IgE-binding to one of the small fragments in more detail, we found further epitope regions on this fragment. It was noteworthy that IgE reactivity to small fragments was weak compared to large fragments or to the complete molecule. Competitive blot inhibition experiments showed that binding of IgE antibodies to the small fragments was specific but with lower avidity than to the complete rHol l 5. rHol l 5 harbours multiple discontinuous as well as continuous IgE-binding epitopes spread over the whole molecule, which were individually recognized by IgE antibodies from different patients. Low avidity of IgE antibodies to small fragments suggests that the continuous epitope regions do not represent the complete epitope and are most probably parts of discontinuous epitopes.     

Expression of the American cockroach Per a 1 allergen in mammalian cells

BACKGROUND: Cockroach allergens are one of the major etiologic risk factors for developing IgE-mediated allergic respiratory illness throughout the world. Per a 1 is a cross-reactive allergen of American and German cockroaches. This study aimed to investigate the expression of a recombinant American cockroach (Periplaneta americana) Per a 1, C42, allergen in mammalian COS-1 cells. METHODS: The COS-1 cells and Escherichia coli were used to express the P. americana C42 allergen. Recombinant proteins were purified with hydroxylapatite and DE52 chromatography. Biologic reactivities of recombinant proteins were examined by direct IgE binding and IgE inhibition studies with the enzyme-linked immunosorbent assay (ELISA). RESULTS: C42 was successfully expressed in the mammalian COS-1 cell as a 50-kDa secreted protein, and purified from the culture medium. The specific human IgE antibodies against recombinant C42 from either E. coli (C42-E. coli) or COS-1 (C42-COS-1) were compared by ELISA with 12 sera from Per a 1 and C42 skin-test-positive patients. All atopic sera contained specific IgE antibodies to C42 from either E. coli or COS-1. Moreover, recombinant C42-COS-1 bound higher levels of serum IgE than recombinant C42-E. coli among C42-sensitive atopic patients, and a statistically significant difference (P<0.01) was found between them. In addition, recombinant C42-COS-1 as an inhibitor revealed higher inhibition of IgE binding to natural Per a 1 than recombinant C42-E. coli. CONCLUSIONS: The biologically highly reactive recombinant C42 produced in the COS-1 cell provides an alternative expression system and will facilitate studies on the immune response of asthma patients to cockroach allergens.