黄唇蜾赢蜂蜂毒中两个过敏原全长基因的克隆、序列分析和表达

为了进一步研究透明质酸酶的过敏活性,利用昆虫杆状病毒成功地表达了黄唇蜾赢蜂Rhynehium brunneum蜂毒的透明质酸酶。根据已报道的胡蜂科透明质酸酶和抗原5基因的氨基酸保守序列,设计合成简并引物,利用反转录多聚酶链式反应（RT—PCR）技术扩增了黄唇蜾赢蜂蜂毒透明质酸酶和抗原5的基因片段,利用RACE技术进一步获得了它们的全长基因（GenBank登录号分别为EU624135和EU624136）。按照过敏原的命名法则,分别命名为Rhyb2和Rhyb5。序列比对分析发现,这两个基因与胡蜂科的相应序列高度相似,说明对胡蜂蜂毒过敏的人群也可能对蜾赢蜂蜂毒有交叉过敏反应。但进一步分析发现,黄唇蜾赢蜂蜂毒透明质酸酶的B细胞决定表位显著不同,9个保守性氨基酸在蜾赢蜂中仅保留了3个,而且缺乏两个关键的精氨酸;黄唇蜾赢蜂蜂毒抗原5序列N端的二级结构和具有过敏活性的常见黄胡蜂Vespula vulgaris蜂毒的抗原5显著不同,有可能因此而缺失依赖其N端二级结构的B细胞决定表位。据此认为,蜾赢蜂蜂毒透明质酸酶和抗原5很可能是天然弱化的过敏原,在过敏原特异性免疫治疗上具有潜在的应用价值。     

米蛾体内Wolbachia的wsp基因序列测定与系统发育分析

Wolbachia是广泛分布于节肢动物体内的一类共生菌, 它们参与多种调控寄主的生殖活动机制。通过对wsp基因的特异性扩增和测序,发现了Wolbachia在米蛾Corcyra cephalonica (Stainton)体内的感染。利用所测序列和其他已发表的序列建立系统树,结果表明米蛾体内Wolbachia属于B大组的Pip类群,与其寄生物茧蜂及赤眼蜂中的Wolbachia各株系遗传距离相差较远。据此推测米蛾体内感染的Wolbachia不是由寄生物（茧蜂、赤眼蜂）水平传播所致。     

家蚕细胞色素P450基因Bmcyp6u1的克隆、序列分析与表达谱

细胞色素P450第6亚家族基因为昆虫所特有,与抗性相关。为了检测家蚕Bombyx mori cyp6u1基因是否与耐氟性相关,首先克隆了cyp6u1基因。采用生物信息学方法获得与黑腹果蝇Drosophila melanogaster cyp6u1基因同源的家蚕B. mori cyp6u1基因序列, 预测该序列的开放阅读框（ORF）为1 476 bp, 编码491个氨基酸, 推定的蛋白质分子质量为56.15 kD, 等电点为9.23。以家蚕5龄第3天幼虫精巢cDNA为模板, 设计特异引物PCR扩增出一条约1 500 bp的条带, 大小与家蚕cyp6u1序列的ORF预测值接近, 命名为Bmcyp6u1基因（GenBank登录号：HM130560）。同源性分析表明, Bmcyp6u1基因与蜜蜂Apis mellifera的同源基因cyp6AS13的相似性为56%, 与拟南芥Arabidopsis thaliana的cyp72A82的相似性为48%, 与人Homo sapiens的cyp3A7基因的相似性为50%。芯片数据分析表明, Bmcyp6u1基因在家蚕5龄第3天幼虫各组织表达量很低, 只在精巢组织(5龄第3天)稍有表达, 推测该基因具有组织特异性。     

苏云金芽孢杆菌Rpp02菌株cry1Ac20基因的克隆及表达

Rpp02菌株是本实验室分离的一株对鳞翅目等多种害虫具有高毒力的苏云金芽孢杆菌莫里逊亚种 (Bacillus thuringiensis subsp. morrisoni), 经PCR检测,它含有cry1Ac基因。对其基因组DNA进行PCR扩增,得到大约4 kb的产物。测序结果表明,该片段含有一个较大的ORF框,基因编码区为3 534 bp,编码1 177个氨基酸,分子量为133.144 kD,pI 4.952, 为弱酸性蛋白质,亮氨酸（Leu）、丝氨酸（Ser）、谷氨酸（Glu）3种氨基酸含量最高,分别为8.0%、7.8%、7.7%。该基因序列与cry1Ac序列同源性达到99%,并被国际Bt杀虫晶体蛋白基因命名委员会命名为cry1Ac20。生物测定表明,该基因在大肠杆菌中得到了表达,表达产物具有较强的杀虫效果,试喂菜青虫48 h后,校正死亡率为88.78%。     

斜纹夜蛾两个天蚕素D基因的克隆及序列分析

天蚕素是昆虫抵御病菌入侵的一类抗菌肽家族。根据斜纹夜蛾Spodoptera litura天蚕素B基因设计特异引物,通过PCR扩增得到2个新的天蚕素基因部分序列,分别命名为cecD1和cecD2（GenBank登录号分别为EF555567和EF555568）。2个基因编码同一个天蚕素D蛋白,该蛋白的成熟肽与天蚕素B存在2个氨基酸残基差异。序列分析发现cecD1和cecD2中分别包含568 bp和377 bp的内含子序列,它们有相同的5′和3′拼接位点,A+T含量分别为59.7%和69.8%,符合大多数真核生物内含子高A+T含量的特征。     

家蚕核型多角体病毒egt基因的分子进化分析

过PCR方法获得家蚕核型多角体病毒（Bombyx mori nuclearpolyhedrosis virus,BmNPV）的蜕皮甾体尿苷二磷酸葡萄糖基转移酶基因（egt）片段,序列分析表明该片段带有EGT的完整ORF,推测的多肽可形成EGT结构域的高级结构。为了研究egt的起源,利用家蚕基因组数据库,电子克隆了多个家蚕尿苷二磷酸葡萄糖醛酸转移酶（UGT）基因,在此基础上进行了进化分析,表明BmNPV的EGT为antennal-enriched型UGT;推测核型多角体病毒（nucleopolyhedrivirus,NPV）和颗粒体病毒（granulovirus,GV）的egt基因在进化上来源于昆虫的UGT基因,但GV的egt基因在进化上的起源可能要早于NPV的egt基因;可能在昆虫祖先种进化形成不同昆虫目的某一时期,杆状病毒的祖先种从昆虫中获得了antennal-enriched型UGT基因,并进化为egt基因。家蚕的部分UGT基因与转座子元件连锁的基因组结构特点反映了杆状病毒的egt基因可能通过转座子的传递而获得。     

感染稻水象甲的Wolbachia基因组中插入序列的鉴定与分析

共生细菌Wolbachia对宿主的生殖起多种调控作用。以往研究表明, Wolbachia基因组中广泛存在插入序列(insertion sequence, IS), 它们对宿主基因组的可塑性、 多样性和进化起重要作用。稻水象甲Lissorhoptrus oryzophilus Kuschel在东亚是一种外来水稻害虫, 在原产地北美营两性生殖, 而在所有入侵地均营孤雌生殖。本研究采用PCR法从河北唐海孤雌生殖型稻水象甲体内克隆获得了Wolbachia的2条IS序列, 即ISWosp4和ISWosp6; 从美国德克萨斯州两性生殖型稻水象甲成虫体内克隆获得了Wolbachia的2条IS序列, 即ISWosp3和ISWosp5。碱基序列比对显示: ISWosp3和ISWosp4属于IS3家族IS3组成员, ISWosp5为IS4家族IS231组成员, ISWosp6为IS5家族IS1031组成员。对这些IS的ORF结构、 所编码氨基酸序列的结构等进行了分析, 推测ISWosp5具有潜在转座活性。所得结果增进了我们对Wolbachia IS3, IS4和IS5家族插入序列的认识, 同时为今后从IS的角度探讨Wolbachia与稻水象甲生殖的关系奠定了基础。     

棉铃虫细胞色素P450 CYP6B7基因的克隆与融合表达

细胞色素P450 CYP6B7被推测与棉铃虫Helicoverpa armigera对拟除虫菊酯类杀虫剂的抗性有关,但至今尚无CYP6B7参与杀虫剂代谢方面的直接证据。为揭示CYP6B7的代谢功能,作者以棉铃虫幼虫基因组DNA 为模板,以CYP6B7基因设计特异性引物,扩增出包含321 bp内含子的CYP6B7基因。用反向PCR的方法消除内含子,获得包含完整的CYP6B7基因的开放阅读框。将CYP6B7基因与pMAL-c2X载体连接,并转化E.coli TB1细胞,在IPTG诱导下,CYP6B7能与载体基因编码的麦芽糖结合蛋白（MBP）在大肠杆菌中融合表达,表达产物经直链淀粉（amylose） 柱亲和层析分离洗脱后,得到SDS-PAGE电泳纯的融合蛋白。     

棉铃虫性染色体两种分子标记的克隆及序列分析

为了建立棉铃虫Helicoverpa armigera性染色体的特异性分子标记,利用RAPD-PCR技术对雌雄棉铃虫基因组DNA进行筛选,从500种随机引物中筛选到1 条引物（Operon编号为AF-18）,可扩增出1条约450 bp 的雌性特异片段。经克隆测序并合成特异引物进行验证,表明该片段为棉铃虫雌性特异分子标记,位于W染色体上。利用家蚕、果蝇等昆虫Kettin基因序列,克隆了棉铃虫的同源基因HaKettin片段,并采用荧光定量PCR技术,以棉铃虫的DH-PBAN基因为参照基因,检测棉铃虫雌雄不同个体间HaKettin基因与DH-PBAN基因的拷贝数之比,结果表明：雄体HaKettin∶DH-PBAN=1.0,雌体HaKettin∶DH-PBAN=0.5,据此推断HaKettin基因位于棉铃虫Z染色体上。     

朱砂叶螨热激蛋白HSP70基因TCHSP70-4的克隆与表达

为了研究朱砂叶螨Tetranychus cinnabarinus(Boisduval)热激蛋白HSP70的表达与其适应高温和低温胁迫的关系,我们利用物种的同源性及RACE 技术,获得朱砂叶螨热激蛋白HSP70基因1个,命名为TCHSP70-4（GenBank 登录号为EU977182）。该基因全长2 182 bp,包含1 959 bp的开放阅读框,编码653个氨基酸,理论分子量为70.9 kDa,等电点为5.4,含有HSP70家族高度保守的基序。运用real-time PCR分析冷激（4℃）和热激（40℃）1 h后TCHSP70-4在朱砂叶螨体内的表达量。结果显示冷激后TCHSP70-4表达量明显下降,而热激后TCHSP70-4表达量却明显上升。这些结果一方面表明该基因属于诱导型HSP70基因,另一方面揭示了朱砂叶螨分别受到冷和热胁迫后体内TCHSP70-4的表达及所起的保护作用是不同的。     

Protein allergens of white-faced hornet, yellow hornet, and yellow jacket venoms.

White-faced hornet, yellow hornet, and yellow jacket venoms have very similar protein compositions; each contains mainly three basic proteins. Two of these proteins have hyaluronidase and phospholipase activities and the third one, designated as antigen 5, is of as yet unidentified biochemical function. These three proteins have molecular weights of about 45 000, 35 000, and 25 000, respectively. The three proteins of white-faced hornet venom have been purified to near homogeneity, while this is the case only for antigen 5 of yellow hornet and yellow jacket venoms. Strong antigenic cross-reaction of the hyaluronidase from these three vespid venoms was observed using specific rabbit anti-venom sera, while weak cross-reactions of phospholipases and of antigen 5s were observed. All three proteins are active as allergens to varying degrees in vespid sensitive individuals. With each vespid venom its antigen 5 seems to be the major allergen. The results help to clarify the commonly observed varying degrees of multiple sensitivity of people to different vespids.     

Characterization of the major allergens purified from the venom of the paper wasp Polistes gallicus

Allergic reactions to vespid stings are one of the major causes of IgE-mediated anaphylaxis. Vespa and Vespula venoms are closely related; Polistes venom is more distantly related and its allergens are less well studied. There is limited cross-reactivity between Polistes and the other vespid venoms because of differences in the epitopes on the allergen molecules.In this study, the major allergens of Polistes gallicus are isolated and characterized. P. gallicus venom contains four major allergens: phospholipase, antigen 5 (Ag5), hyaluronidase and protease that were characterized by mass spectrometry and specific binding to IgE. The complete amino acid sequence of Ag5 and the sequence of the N-terminal region of phospholipase were also determined. The alignment of Ag5 from P. gallicus (European species) and Polistes annularis (American species) shows an 85% identity that increases to 98% within the same subgenus. This could suggest the presence of specific epitopes on Ag5 molecule being the variations on the superficial loops. The features of the P. gallicus allergens could explain the partial cross-reactivity found between the American and European Polistes venoms, and suggest that the use of European Polistes venoms would improve the diagnostic specificity and the therapy of European patients and of North American patients sensitized by European Polistes.     

Isolation, cloning and characterization of Polistes dominulus venom phospholipase A1 and its isoforms

Polistes dominulus is known to be an allergenically important social wasp. Its venom has four major allergens (ziv. phospholipase A1, hyaluronidase, antigen 5 and a serine-protease). Amino acid sequences of its serine-protease and Antigen 5 have been published. In this paper, the partial amino acid sequence of its venom phospholipase native protein is reported. Also, we give an account to the complete nucleotide sequence of Polistes dominulus venom phospholipase A1 gene, its isoforms and their complete deduced amino acid sequences. Their similarity to the other phospholipases A1 of the family: Vespidae is discussed.     

Molecular cloning, recombinant expression and IgE-binding epitope of omega-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis

Wheat omega-5 gliadin has been identified as a major allergen in wheat-dependent exercise-induced anaphylaxis. We have detected seven IgE-binding epitopes in primary sequence of the protein. We newly identified four additional IgE-binding epitope sequences, QQFHQQQ, QSPEQQQ, YQQYPQQ and QQPPQQ, in three patients with wheat-dependent exercise-induced anaphylaxis in this study. Diagnosis and therapy of food allergy would benefit from the availability of defined recombinant allergens. However, because omega-5 gliadin gene has not been cloned, recombinant protein is currently unavailable. We sought to clone the omega-5 gliadin gene and produce the homogeneous recombinant protein for use in an in vitro diagnostic tool. Using a PCR-based strategy we isolated two full-length omega-5 gliadin genes, designated omega-5 and omega-5b, from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by omega-5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa; the omega-5b gene would encode a 393 amino acid, but it contains two stop codons indicating that omega-5b is pseudogene. The C-terminal half (178 amino acids) of the omega-5a gliadin protein, including all 11 IgE-binding epitope sequences, was expressed in Escherichia coli by means of the pET system and purified using RP-HPLC. Western blot analysis and dot blot inhibition assay of recombinant and native omega-5 gliadin purified from wheat flour demonstrated that recombinant protein had IgE-binding ability. Our results suggest that the recombinant protein can be a useful tool for identifying patients with wheat-dependent exercise-induced anaphylaxis in vitro.     

Allergens of honey bee venom.

The allergenic activities of four purified components of honeybee venom were studied by using histamine release from leukocytes of bee sting-allergic patients. The components studied were hyaluronidase, phospholipase A₂, melittin and apamin with molecular weights, respectively, of about 50,000, 15,800, 2840 and 2038 d. In six of the seven patients studied, hyaluronidase and phospholipase were, respectively, on the average about two and eight times more active by weight than the venom. The situation was reversed in one patient in that hyaluronidase and phospholipase A₂ were, respectively, 90 and 0.5 times more active than the venom. With this single exception, hyaluronidase and phospholipase were about equally active on a molar basis as allergens. Melittin was on the average about one-tenth as active as the venom, and apamin was inactive as an allergen.Chemical modifications of phospholipase A₂ were carried out. Succinylation of eight of its eleven amino groups yielded a derivative that retained 4% of the enzymic activity of the native enzyme. Reduction and carboxymethylation of its four disulfide bonds or cyanogen bromide cleavage of its three methionyl bonds yielded enzymatically inactive derivatives. These derivatives showed varying decreases of allergenic activities when compared to the native enzyme. The results indicate that the antigenic determinants of phospholipase depend on the charge, the amino acid sequence and the conformation of the molecule.     

Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab

The major allergens of honeybee venom, hyaluronidase (Hyal) and phospholipase A2, can induce life-threatening IgE-mediated allergic reactions in humans. Although conventional immunotherapy is effective, up to 40% of patients develop allergic side effects including anaphylaxis and thus, there is a need for an improved immunotherapy. A murine monoclonal anti-Hyal IgG1 antibody (mAb 21E11), that competed for Hyal binding with IgEs from sera of bee venom allergic patients, was raised. The fragment of these IgG antibodies which bind to antigen (Fab) was produced and complexed (1:1) with Hyal. The crystal structure determination of Hyal/Fab 21E11 complex (2.6 A) enabled the identification of the Hyal-IgG interface which provides indirect information on the Hyal-IgE interaction (B-cell epitope). The epitope is composed of a linear array of nine residues (Arg138, His141-Arg148) located at the tip of a helix-turn-helix motive which protrudes away from the globular core and fits tightly into the deep surface pocket formed by the residues from the six complementarity determining regions (CDRs) of the Fab. The epitope is continuous and yet its conformation appears to be essential for Ab recognition, since the synthetic 15-mer peptide comprising the entire epitope (Arg138-Glu152) is neither recognized by mAb 21E11 nor by human IgEs. The structure of the complex provides the basis for the rational design of Hyal derivatives with reduced allergenic activity, which could be used in the development of safer allergen-specific immunotherapy.     

A proteomic study of the major allergens from yellow jacket venoms

The venoms of stinging insects belong to the most dangerous allergen sources and can cause fatal anaphylactic reactions. Reliable prediction of a patient's risk to anaphylactic reactions is vital, and diagnosis requires the knowledge of the relevant allergens. Recently, a new hyaluronidase -like glycoprotein from Vespula vulgaris (Ves v 2b) was identified. This led us to investigate hyaluronidases and also other major allergens from V. germanica and four additional Vespula species. By MALDI-Q-TOF-MS, the new hyaluronidase-like protein was shown to be the major component of the 43-kDa band in all Vespula species studied. LC-ESI-Q-TOF-MS/MS sequencing of Ves g 2a and Ves g 2b facilitated the cloning of their cDNA. Ves v 2b and Ves g 2b turned out to be essentially identical on protein level. Whereas the less abundant "a" form displayed enzymatic activity, the new "b" homologue did not. This is probably caused by amino acid exchanges in the active site, and it raises questions about the physiological role of this protein. Sequence comparisons by MS/MS of antigen 5 and phospholipases from V. vulgaris, germanica, maculifrons, pensylvanica, flavopilosa and squamosa revealed the latter as a taxonomic outlier and led to the discovery of several not previously reported amino acid differences.     

Characterization of Pen n 13, a major allergen from the mold Penicillium notatum

Penicillium notatum is a well-known indoor aeroallergen and is frequently included in skin test panels for allergic diagnosis. On two-dimensional immunoblotting using patients' sera containing IgE and monoclonal antibody D7B8 specific for Pen c 1 of P. citrinum, two allergens with a molecular mass of 33 kDa but different isoelectric points were identified. A novel cDNA coding for Pen n 13 was cloned and sequenced. The nucleotide sequence codes for a protein 397 amino acids including a putative signal peptide of 25 amino acids and a propeptide of 90 amino acids. The allergen is an alkaline serine protease that shares more than 39% identical residues with other kinds of mold allergens. The coding cDNA of Pen n 13 was cloned into vector pQE-30 and expressed in E. coli M15 as a His-tag fusion protein and purified to homogeneity. The fusion protein reacted with monoclonal antibodies of Pen c 1 and with IgE from Penicillium-allergic patients. Furthermore, it also cross-reacted strongly with IgE specific for the natural Pen c 1, indicating that similar IgE binding epitopes may exist in the allergens of P. notatum and P. citrinum. Antigenicity index plots indicated that there are several similar epitope regions of high antigenic indices in Pen c 1 and Pen n 13, corroborating that mold allergens belonging to the alkaline serine protease family possess similar protein structure and strong antigenic cross-reactivity.     

Hypervariable region IV of Salmonella gene fliCd encodes a dominant surface epitope and a stabilizing factor for functional flagella.

To identify the major antigenic determinant of native Salmonella flagella of antigenic type d, we constructed a series of mutated fliCd genes with deletions and amino acid alterations in hypervariable region IV and in region of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T.M. Joys and F. Schödel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the mutant genes, with deletions of up to 92 amino acids in region IV, assembled into functional flagella and conferred motility on flagellin-deficient hosts. Serological analysis of these flagella with different anti-d antibodies revealed that the peptide sequence centered at amino acids 229 to 230 of flagellin was a dominant B-cell epitope at the surface of d flagella, because replacement of these two amino acids alone or together with their flanking sequence by a tripeptide specified by a linker sequence eliminated most reactivity with antisera against wild-type d flagella as tested by enzyme-linked immunosorbent assay or by Western immunoblot. Functional analysis of the mutated flagellin genes with or without an insert suggested that amino acids 180 to 214 in the 5' part of hypervariable region IV (residues 181 to 307 of the total of 505) is important to the function of flagella. The hybrid proteins formed by insertion of peptide sequence pre-S1 12-47 of hepatitis B virus surface antigen into the deleted flagellins assembled into functional flagella, and antibody to the pre-S1 sequence was detected after immunization of mice with the hybrid protein. This suggests that such mutant flagellins containing heterologous epitopes have potential as vaccines.