尘螨变应原Derf1核酸序列测定及其分子进化分析

目的:对尘螨主要变应原Der f1进行核酸序列测定,探讨其系统进化信息。方法:根据Genbank公布的Der f1基因序列设计引物,巢式PCR扩增Der f1的cDNA,纯化、回收、克隆至pMD19-T simple后进行序列测定,序列比对后用Clustal W 1.83构建分子进化树。结果:成功扩增出Der f1的cDNA片段,测序表明该基因含ORF1个,长度966bp,与参考序列同源性达99.9%。该变应原具半胱氨酸蛋白酶活性,与果蝇进化关系最远,与梅氏嗜霉螨进化关系最近。结论:成功获得了尘螨变应原Der f1基因片段,根据其编码的氨基酸序列构建出的系统进化树与形态学分类不一致。     

尘螨变应原Der f1全序列的原核表达及生物信息学分析

目的 构建尘螨变应原Der f1原核表达体系,并了解其分子特征.方法 提取粉尘螨总RNA,用RT-PCR合成Der f1编码基因,将其克隆至pMD19-T载体,亚克隆至表达载体pET-28a( ),转化至大肠杆菌并用IPTG诱导表达.用生物信息学软件对测序结果进行分析并预测其空间结构.结果 从粉尘螨总RNA中扩增获得Der f1 cDNA片段,成功构建了表达质粒pET-28a( )-Der f1,Western blotting显示原核表达获得成功.测序结果提交GenBank,登陆号为EU095368,该基因长966 bp,与参考序列同源性达99.9%,推测其编码氨基酸321个,属疏水蛋白,位于细胞外,信号肽位于1～18氨基酸处.同源性分析提示Der f1和Eur m1相似率为88%,而Der f1和Der p1的相似率为77%,分子进化树中粉尘螨和梅氏嗜霉螨聚成一簇.Der f1的二级结构由α-螺旋(109 aa,33.96%)、延伸链(55 aa,17.13%)、β-转角(18 aa,5.61%) 和随机卷曲(139 aa,43.30%)组成.结论 尘螨变应原Der f1原核表达获得成功,为进一步生产重组变应原奠定了基础.生物信息学分析表明粉尘螨和梅氏嗜霉螨的亲缘关系可能更近,而与屋尘螨关系稍远,此与现行的形态学分类系统并不符合.     

尘螨变应原Der fl全序列的原核表达及生物信息学分析

目的 构建尘螨变应原Der fl原核表达体系,并了解其分子特征。方法 提取粉尘螨总RNA,用RT—PCR合成Der fl编码基因,将其克隆至pMD19-T载体,亚克隆至表达载体pET-28a（＋）,转化至大肠杆菌并刚IPTG诱导表达。用生物信息学软件对测序结果进行分析并预测其空间结构。结果从粉尘螨总RNA中扩增获得Der fl cDNA片段,成功构建了表达质粒pET-28a（＋）-Der fl,Western blotting显示原核表达获得成功。测序结果提交GenBank,臀陆号为EU095368,该基因长966bp,与参考序列同源性达99．9％,推测其编码氩基酸321个,属疏水蛋白,位于细胞外,信号肽位于1～18氨基酸处。同源性分析提示Der fl和Eur ml相似率为88％,而Der fl和Derpl的相似率为77％,分子进化树中粉尘螨和梅氏嗜霉螨聚成一簇。Derfl的二级结构由α-螺旋（109aa,33．96％）、延伸链（55aa,17．13％）、β-转角（18aa,5．61％）和随机卷曲（139aa,43．30％）组成：结论 尘螨变麻原Der fl原核表达获得成功,为进一步生产重组变麻原奠定了基础。生物信息学分析表明粉尘螨和梅氏嗜霉螨的亲缘关系可能更近,而与屋尘螨关系稍远,此与现行的形态学分类系统并不符合。     

尘螨变应原Der f1植物表达产物诱导哮喘小鼠免疫耐受的研究

目的:采用尘螨变应原组分Der f1植物表达产物免疫治疗哮喘小鼠,了解其诱导哮喘小鼠免疫耐受的效果及机制.方法:将BALB/c小鼠分为正常组、哮喘组和治疗组,治疗组在致敏后分别给予尘螨变应原Der f1原核表达产物(rDE)和烟草叶片中的表达产物(rDP)免疫治疗,处死小鼠,取支气管肺泡灌洗液(BALF)、血清和肺组织,分别进行细胞学、螨特异性抗体、细胞因子和组织病理学检查,观察这些指标的变化.结果:哮喘组和治疗组BALF中细胞总数明显增多,中性和嗜酸性粒细胞超过50%;rDE和rDP治疗后,小鼠BALF中细胞总数和嗜酸性粒细胞减少;哮喘组和治疗组小鼠螨特异性IgE、IgG和IL-4水平升高,IFN-γ水平下降(P<0.01);rDE和rDP治疗后,IgE、IgG和IL-4水平下降,IFN-γ水平上升(P<0.01-0.05),以rDP疗效更好;哮喘组小鼠支气管、细支气管和小血管周围可见明显炎性细胞浸润,rDE和rDP治疗后,炎症减轻,以rDP改变更为明显.结论:尘螨变应原Der f1植物表述产物较原核表达产物能更有效地减轻螨性哮喘小鼠的气道炎症,诱导免疫耐受形成.     

尘螨变应原Der f1 真核表达载体的构建及转染CHO 细胞

目的:构建尘螨变应原Der f1真核表达载体,转染真核细胞并进行蛋白表达.方法:根据Genebank中Der f1基因的核酸序列(AB034946),设计引物,采用PCR法,从保存的JM109工程菌中扩增Der f1编码基因,克隆到真核表达质粒pcDNA3.1/myc-hisA上,以脂质体法转染CHO细胞,经G418筛选,进行稳定表达细胞株的筛选和鉴定.结果:将目的基因Der f1成功连接到pcDNA3.1/myc-hisA-Derf1并转染CHO细胞,获得稳定表达的CHO细胞株.结论:成功构建了尘螨变应原Der f1真核表达载体,并转染CHO细胞表达蛋白质.     

尘螨变应原Der f 9、Der p 9和Blo t 9结构与功能的比较研究

使用多种生物信息学工具来预测、比较尘螨变应原Der f 9、Der p 9和Blo t 9的一级、二级、三级结构及抗原表位,找出3种蛋白结构及功能的异同。Der f 9、Der p 9和Blo t 9氨基酸序列一致性为82.07%,都含有3个胰蛋白酶活性位点氨基酸及2个功能结构区特异性序列;二级和三级结构中都由α-螺旋、β-折叠和无规卷曲组成;活性位点氨基酸在三级结构中完全重合,并相互靠近构成蛋白酶的活性中心;主要潜在抗原表位区域都为5个,并在第47-49aa和200-203aa区域出现了重合,但表位重合区的氨基酸种类不完全相同。应用生物信息方法预测和比较了Der f 9、Der p 9和Blo t 9结构与抗原方面的信息,为进一步研究变应原第9组分的生物学功能、疫苗研制奠定了基础。     

屋尘螨Ⅰ类变应原Der p1的体内定位

对过敏性疾病主要过敏原屋尘螨Dermatophagoides pteronyssinusⅠ类变应原（Der p1）进行了抗原定位研究。选用经表达和纯化的Der p1抗原蛋白,按常规方法免疫小鼠,分离血清获抗重组Der p1的抗体（Ⅰ抗）,用抗鼠IgG荧光抗体为Ⅱ抗,屋尘螨经石蜡切片,在荧光显微镜下对其特异性变应原的定位进行观察。HE染色显示屋尘螨消化系统占据大部分体腔。组织免疫荧光发现屋尘螨Ⅰ类变应原主要定位于螨的中肠组织及肠内容物,而螨的生殖系统、排泄系统等脏器以及几丁质甲壳均呈阴性反应。据此认为屋尘螨Ⅰ类变应原存在于螨的肠内容物和中肠组织中。     

尘螨变应原Der p1浓度与哮喘患者血清螨特异性抗体的季节消长

目的:探讨哮喘患者血清抗体及患者室内尘螨抗原浓度的季节变化规律。方法:2005年9月、2005年12月、2006年3月、2006年6月用ELISA法检测哮喘患者卧室尘样中Der P 1浓度,同步检测患者外周血总IgE、s-IgE、s-IgG1、s-IgG2和s-IgG4。结果:哮喘患者血清总IgE、s-IgE、s-IgG1、s-IgG2和s-IgG4均高于正常对照组,差异均具显著性(P<0.01);比较患者血清总IgE、s-IgE及s-IgG1、s-IgG2、s-IgG4四次检测值,差异均具显著性(P<0.01),患者血清总IgE、s-IgE在12月份检测值最高、6月份检测值最低,s-IgG1以6月份最高、3月份最低,s-IgG4在3月份最高、6月份最低。统计表明,患者卧室Der P1浓度四次检测值差异具有显著性(P<0.01),以2006年3月和2005年12月为高。结论:哮喘患者血清总IgE、s-IgE、s-IgC1、s-IgG2和s-IgG4和卧室尘螨抗原浓度呈季节变化,IgG1的变化与尘螨抗原浓度的变化趋势相反,IgG4的变化与尘螨抗原浓度的变化趋势一致。     

药用野生稻复合体ITS1和ITS2序列变异及其系统进化分析

通过PCR扩增并测序分析稻属药用野生稻复合体5个野生稻种基因组完整的ITS区及5.8S区,并与栽培稻ITS序列进行比较,构建分子系统进化树,探讨了稻属药用野生稻复合体内不同种间的亲缘关系和系统进化.结果表明,ITS1和ITS2均有较高的G/C含量,ITS1序列的长度多态性相对较高,ITS2序列的碱基突变频率较高.药用野生稻和高秆野生稻亲缘关系很近,而与栽培稻亲缘关系较远;短药野生稻、斑点野生稻、澳洲野生稻与药用野生稻亲缘关系渐近.处于进化的过渡阶段.     

分子进化生物学中序列分析方法的新进展

简要介绍了分子进化生物学中序列分析方法的最新进展,特别强调了似然比检验和贝叶斯推论在分子进化和系统发育假说检验中的重要性,并介绍了新方法的一些成功应用,同时还给出了一些重要的信息资源。     

粉尘螨过敏原蛋白Der f 3 的表达纯化及活性鉴定

目的:制备有生物学活性的粉尘螨过敏原蛋白Der f 3。方法:构建带有StrepⅡ标签的原核表达体系Rosetta-p ET44a-Der f 3高效表达目的蛋白,分别用梯度透析法和亲和层析法对其进行复性纯化,通过Western Blot试验验证目的蛋白的有效表达,最后用丝氨酸蛋白酶特异荧光底物及粉尘螨过敏病人血清分别鉴定目的蛋白的酶学活性及免疫学活性。结果:利用原核表达体系成功表达了目的蛋白Der f 3,Western Blot结果显示有目的条带出现。复性纯化后的过敏原蛋白Der f 3能够成功降解丝氨酸蛋白酶特异性荧光底物,ELISA试验结果显示该蛋白血清特异性Ig E在4级以上粉尘螨过敏病人中阳性率为4%。结论:成功获得有酶学活性及免疫学活性的粉尘螨过敏原蛋白Der f 3,为生产高质量的诊断试剂和疫苗奠定了基础。     

Recombinant Der p 1 and Der f 1 exhibit cysteine protease activity but no serine protease activity

Although mite major group 1 allergens, Der p 1 and Der f 1, were first isolated as cysteine proteases, some studies reported that natural Der p 1 exhibits mixed cysteine and serine protease activity. Clarifying whether the serine protease activity originates from Der p 1 or is due to contamination is important for distinguishing between the pathogenic proteolytic activities of group 1 allergens and mite-derived serine proteases. Recombinant mite group 1 allergens would be useful tool for addressing this issue, because they are completely free from contamination by mite serine proteases. Recombinant Der p 1 and Der f 1, and highly purified natural forms exhibited only cysteine protease activity. However, commercially available natural forms exhibited both activities, but the two activities were eluted into different fractions in size-exclusion column chromatography. The substrate specificity associated with the serine protease activity was similar to that of Der f 3. These results indicate that the serine protease activity does not originate from group 1 allergens.     

Modelling and Bioinformatics Analysis of the Dimeric Structure of House Dust Mite Allergens from Families 5 and 21: Der f 5 Could Dimerize as Der p 5

Abstract

Allergy represents an increasing thread to public health in both developed and emerging countries and the dust mites Dermatophagoides pteronyssinus (Der p), Blomia tropicalis (Blo t), Dermatophagoides farinae (Der f), Lepidoglyphus destructor (Lep d) and Suidasia medanensis (Sui m) strongly contribute to this problem. Their allergens are classified in several families among which families 5 and 21 which are the subject of this work. Indeed, their biological function as well as the mechanism or epitopes by which they are contributing to the allergic response remain unknown and their tridimensional structures have not been resolved experimentally except for Blo t 5 and Der p 5. Blo t 5 is a monomeric three helical bundle, whereas Der p 5 shows a three helical bundle with a kinked N-terminal helix that assembles in an entangled dimeric structure with a large hydrophobic cavity. This cavity could be involved in the binding of hydrophobic ligands, which in turn could be responsible for the shift of the immune response from tolerance to allergic inflammation. We used molecular modelling approaches to bring out if other house dust mite allergens of families 5 and 21 (Der f 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) could dimerize and form a large cavity in the same way as Der p 5. Monomeric models were first performed with MODELLER using the experimental structures of Der p 5 and Blo t 5 as templates. The ClusPro server processed the selected monomers in order to assess their capacity to form dimeric structures with a positive result for Der p 5 and Der f 5 only. The other allergens (Blo t 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) did not present such a propensity. Moreover, we identified mutations that should destabilize and/or prevent the formation of the Der p 5 dimeric structure. The production of these mutated proteins could help us to understand the role of the dimerization process in the allergic response induced by Der p 5, and if Der p 5 and Der f 5 behave similarly.     

House dust mite allergen Der f 1 induces IL-8 in human basophilic cells via ROS-ERK and p38 signal pathways

Der f 1, a major house dust mite allergen and member of the papain-like cysteine protease family, can provoke immune responses with its proteolytic activity. To understand the role of Der f 1 in inflammatory immune responses, we studied the mechanism of the regulation of interleukin (IL)-8 expressions in human basophilic cell KU812 by proteolytically active recombinant Der f 1. Not only production of IL-8 mRNA was induced but also the DNA binding activity of activator protein-1 (AP-1) and phosphorylation of NF-κB p65 were increased in Der f 1-treated KU812. Furthermore, Der f 1 induction of IL-8 expression was sensitive to pharmacological inhibition of ERK and p38 mitogen activated protein kinase (MAPK) pathways. Der f 1 also activated ERK and p38 MAPK phosphorylation and rapidly induced reactive oxygen species (ROS) production. The antioxidant N-acetyl-cysteine (NAC) inhibited phosphorylation of ERK, but not p38, suggesting that secretion of IL-8 in KU812 cells treated with Der f 1 is dependent on ROS, ERK MAPK and p38 MAPK. We describe the mechanism of Der f 1-induced IL-8 secretion from human basophilic cells, which are thought to be important for allergic inflammation independent of IgE antibodies. These findings improve our understanding of the inflammatory immune response in human basophils to protease allergens.     

粉尘螨变应原第7组分全长基因序列测定与分析

目的：获得粉尘螨变应原第7组分编码基因并了解其分子特征。方法：根据GenBank已公布的Derf7核酸序列设计引物,用RT—PCR扩增获得其编码基因,插入pMD19-T载体进行序列测定和生物信息学分析。结果：获得Derf7全长基因约642bp,与参考序列（GenBankAY283292）同源性达99．7％％,仅在249位”A→G”和439位“C→T”发生点突变,含1个完整的开放读码框。推测编码蛋白由213个氨基酸组成,信号肽序列位于1-17aa,亲水性指数为0．031,跨膜区域位于171—190aa,二级结构由一螺旋（57．28％）、延伸主链（6．57％）和无规卷曲（36．15％）组成;亚细胞定位于细胞质,含有N-糖基化位点1个（151-154aa）,蛋白激酶c磷酸化位点1个（193-195aa）,酪蛋白激酶Ⅱ磷酸化位点2个（155—158aaand173．176aa）。N端酰基化位点1个（97—102aa）。粉尘螨和屋尘螨变应原第7组分氨基酸序列相似度为86％,二者在螨类第7组分氨基酸序列构建出的分子进化树中聚成一簇。结论：获得了Derf7全长基因,为进一步获得其基因工程制品用于临床和实验研究奠定了基础。     

屋尘螨致敏/激发小鼠气道变态反应性炎症模型的构建

目的使用屋尘螨(HDM)提取液致敏和激发C57BL/6小鼠构建气道变态反应性炎症模型的方法。方法模型组和对照组,各8只。模型组以HDM致敏和激发小鼠,分别于第0、7、14、21天腹腔注射HDM,10 d后,连续雾化吸入HDM 3 d。对照组以PBS代替HDM。进行支气管肺泡灌洗液(BALF)细胞总数计数和分类计数,肺组织病理检查,ELISA测定BALF上清中IL-4、IL-5和IFNγ-水平。结果模型组可见明显炎性细胞浸润,以嗜酸性粒细胞为主。而对照组未见明显炎性细胞浸润。BALF中细胞总数计数和嗜酸性粒细胞比例较对照组明显升高(P<0.01);BALF上清中IL-4、IL-5水平较对照组明显升高(P<0.001),IFNγ-水平较对照组降低(P<0.01)。结论使用HDM致敏和激发C57BL/6小鼠成功地建立了小鼠气道变应性炎症模型。     

特异性免疫治疗对屋尘螨致敏哮喘小鼠NKT细胞的影响

目的:研究特异性免疫治疗(SIT)对哮喘小鼠自然杀伤T(NKT)细胞的影响。方法:24只BALB/C小鼠随机分为对照组(A组)、哮喘模型组(B组)、哮喘免疫治疗(SIT)组(C组),各8只。通过屋尘螨提取液(HDM)诱导建立哮喘小鼠模型并进行SIT治疗。检测各组小鼠的气道反应性、支气管肺泡灌洗液(BAlF)细胞计数及分类、ELISA检测IL-4、IFN-γ以及应用流式细胞仪检测NKT细胞数目,通过RT-PCR方法检测T-bet和GATA-3mRNA表达水平;HE染色观察小鼠肺组织的改变。结果:与B组相比,C组气道反应性明显下降(P0.01);BALF中细胞总数及嗜酸性粒细胞(EOS)数显著减少(P0.01);血清IL-4分泌显著降低(P0.01),IFN-γ显著升高(P0.01);NKT细胞数及其成熟型比例明显升高(P0.05);T-betmRNA表达水平明显升高(P0.01),且与NKT细胞数及其成熟型比例呈正相关性,GATA-3mRNA表达水平明显降低(P0.05),且与NKT细胞数及其成熟型比例呈负相关性。B组肺部管腔周围炎性细胞聚集,组织上皮损伤,组织水肿,而C组肺部变应性炎症明显减轻。C组其他各项指标接近A组。结论:哮喘的发生可能与NKT细胞失调相关,通过改变NKT细胞数目及其成熟型比例来调节GATA-3/T-bet的表达可能是SIT治疗哮喘的作用机制之一。     

Crystallization and Preliminary X-Ray Diffraction Analysis of Group 5 Mite Allergen fromDermatophagoides pteronyssinus

Crystals of the 14-kDa group 5 allergen fromDermatophagoides pteronyssinus(Der p 5) have been obtained at low pH and diffract to 3-Å resolution using a conventional x-ray source. The crystals belong to tetragonal space group P4₁2₁2 or P4₃2₁2, with unit cell parametersa=b= 114 Å andc= 234 Å. A self-rotation search revealed a 432 point symmetry and thus suggested 96 molecules in one unit cell, hence 12 monomers in each asymmetric unit.     

MHC Class II-Restricted Presentation of the Major House Dust Mite Allergen Der p 1 Is GILT-Dependent: Implications for Allergic Asthma

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is known to reduce disulfide bonds present in proteins internalized by antigen presenting cells, facilitating optimal processing and presentation of peptides on Major Histocompatibility Complex class II molecules, as well as the subsequent activation of CD4-positive T lymphocytes. Here, we show that GILT is required for class II-restricted processing and presentation of immunodominant epitopes from the major house dust mite allergen Der p 1. In the absence of GILT, CD4-positive T cell responses to Der p 1 are significantly reduced, resulting in mitigated allergic airway inflammation in response to Der p 1 and house dust mite extracts in a murine model of asthma.