细粒棘球绦虫EgA31重组抗原与其它寄生蠕虫的免疫交叉反应

为了研究细粒棘球绦虫EgA31重组蛋白的抗原性 ,作者将细粒棘球绦虫 6 6kDa抗原的cDNA克隆EgA31 3′端 5 0 0bp碱基片断亚克隆入pGEX 5X表达质粒 ,构建EgA31 GST重组蛋白原核表达系统 ,所制备的重组蛋白免疫豚鼠获得抗血清 ;免疫试验表明该抗血清可识别EgA31 GST重组蛋白及细粒棘球绦虫 6 6kDa抗原 ,也可与多房棘球蚴、犬复孔绦虫、犬钩虫、日本血吸虫的成虫虫体总蛋白 6 6kDa抗原分子发生交叉反应 ,提示EgA31抗原分子在上述虫体中均有存在     

miRNA作为骨棘球蚴病诊断标志物的研究进展

骨棘球蚴病通常由细粒棘球绦虫和多房棘球绦虫引起,人类在食用被虫卵污染的食物或水时被感染,骨棘球蚴病的治疗一般包括手术和药物治疗,但治疗时间长、费用高,给患者造成了沉重负担。微小RNA(miRNA)已知参与多种生物过程和宿主-寄主相互作用,包括发育、细胞生长和死亡、寿命的相关靶点调节、转录、信号转导和细胞运动,这将有助于研究人员找到治疗和控制骨棘球蚴病的新策略和靶点。为进一步了解骨棘球蚴病,认清棘球绦虫在最终宿主和中间宿主中发育过程的分子基础至关重要,在细粒棘球绦虫、多房棘球绦虫中发现的miRNA在其各自宿主的表达调控中具有基因和发育阶段特异性。主要对miRNA作为骨棘球蚴病诊断标志物的研究进展进行综述。     

额敏县158例肝包虫手术患者调查

棘球蚴病俗称包虫病,是因棘球绦虫的幼虫寄生于人体组织而引起的人兽共患性寄生虫病,分布于全世界广大的畜牧地区,在人与动物之间传播.在我国多流行于西北和西南牧区.目前已确认的棘球绦虫有4种,即细粒棘球绦虫、多房棘球绦虫、伏氏棘球绦虫及少节棘球绦虫.在我国主要为细粒棘球蚴病和泡型棘球蚴病.据调查,人肝包虫的感染率在3.1~31.5%,忠病率在0.5~5.0%[1]仅新疆每年因患包虫病接受手术者达2000人次以上.已成为农牧民因病致贫和因病反贫的主要原因.严重危害人类的健康.现将我县2004年至2009年158例肝包虫手术患者作如下分析:     

小鼠外周血淋巴细胞亚群在细粒棘球蚴包虫致敏反应中的作用

目的:建立细粒棘球蚴过敏反应BALB/c小鼠模型,研究和探讨淋巴细胞亚群在细粒棘球蚴致敏反应中的作用。方法:从自然感染细粒棘球蚴的绵羊肝脏中提取原头蚴,培养40 d后,以50个微囊/鼠的剂量,通过腹腔注射接种BALB/c小鼠,对照组注射无菌生理盐水。感染6个月之后,每只小鼠按0.1 ml/10 g经腹腔注射羊源细粒棘球...     

细粒棘球绦虫重组质粒pGEX-Eg95的构建及其在大肠杆菌BL21（DE3）中的表达

目的构建细粒棘球绦虫重组质粒pGEX-Eg95,并研究该质粒在大肠杆菌BL21（DE3）中的表达。方法超声粉碎细粒棘球蚴组织提取总RNA,通过RT—PCR扩增Eg95抗原编码基因;克隆至原核表达载体pGEX—1λT,构建重组质粒pGEX-Eg95;转化大肠杆菌BL21,经异丙基硫代-β—D-半乳糖苷（IPTG）诱导表达后用SDS-PAGE和Western blowing对表达产物进行分析和鉴定。结果RT-PCR扩增出471bp的Eg95抗原编码基因;双酶切证实Eg95抗原编码基因成功插入pGEX-1λT中;SDS-PAGE分析显示表达产物为相对分子质量约42500的重组蛋白,与预期结果一致,表达的蛋白约占菌体总蛋白的21％;Western blot鉴定显示重组蛋白能被细粒棘球蚴感染鼠血清识别。结论成功构建了细粒棘球绦虫重组质粒pGEX-Eg95,该质粒在大肠杆菌BL21中获得了高效融合表达,表达的融合蛋白具有特异的抗原性。     

细粒棘球绦虫重组BCG-Eg95疫苗的构建及其表达效率研究

目的 构建细粒棘球绦虫重组BCG-Eg95疫苗,分析Eg95分子在该疫苗中的表达效率.方法超声粉碎细粒棘球蚴组织提取总RNA,通过RT-PCR扩增Eg95的抗原编码基因;将该基因定向克隆到大肠杆菌-分枝杆菌穿梭表达载体pB-CG,构建重组质粒pBCG-Eg95;电穿孔法转化BCG,构建细粒棘球绦虫重组BCG-Eg95疫苗.免疫印迹分析重组BCG-Eg95疫苗的表达产物.结果 RT-PCR成功扩增出471 bp的Eg95抗原编码基因;双酶切证实Eg95抗原编码基因成功插入pBCG中;PCR证实rBCG-Eg95疫苗构建成功;免疫印迹分析发现重组BCG-Eg95疫苗的表达产物在相对分子质量(Mr)约为16.5×103处有明显的目的 蛋白表达条带,且能被感染细粒棘球蚴的鼠血清特异识别.结论 成功构建了细粒棘球绦虫重组BCG-Eg95疫苗,为而后的开发利用奠定了理论基础.     

细胞蜡块在诊断棘球蚴病中的应用北大核心CSCD

棘球蚴病又名包虫病,是细粒棘球绦虫幼虫（棘球蚴）寄生于家畜等多种食草动物和人的组织器官内的人兽共患寄生虫病,多发于世界各地牧区。以往该病的诊断是基于普通细胞学,如今,随着细胞蜡块技术的不断发展,大大拓展了细胞病理学诊断的实用范围和能力。     

细粒棘球绦虫疫苗候选分子EgA31的GST融合蛋白原核表达及纯化

目的 :构建细粒棘球绦虫疫苗候选分子EgA31重组质粒 ,表达及纯化EgA31 GST融合蛋白 ,为疫苗的研究奠定基础。方法 :PCR扩增EgA31cDNA ,将得到的cDNA经限制性内切酶酶切 ,然后亚克隆入pGEX 5X 3质粒 ,转化BL2 1宿主菌 ,IPTG诱导重组质粒的表达 ,亲和层析纯化表达产物 ,Bradford法测定重组蛋白含量 ,用SDS PAGE和Westernblot进行分析鉴定。结果 :SDS PAGE显示分离纯化的EgA31 GST融合蛋白为 4 5kDa ,测序检验重组质粒中EgA31cDNA序列正确。EgA31 GST融合蛋白免疫豚鼠得到的抗血清可与细粒棘球绦虫原头蚴总蛋白在 6 6kDa处特异性反应。结论 :EgA31 GST融合蛋白获得高效表达 ,并成功纯化 ,初步实验证明具有良好的免疫原性 ,可用于进一步疫苗的免疫注射实验     

原头蚴及囊液促进小鼠脾细胞产生IL-2

目的观察细粒棘球绦虫幼虫原头蚴及囊液对体外培养小鼠脾细胞产生IL-22的影响。方法取Balb/c小鼠脾细胞,分别加入不同浓度原头蚴或囊液共培养48 h,检测上清液IL-22表达量及细胞IL-22 m RNA的相对表达量。同浓度原头蚴或囊液分别在0、12、24、36和48 h收集细胞,流式细胞术检测CD4+IL-22+T细胞比例变化。RPMI 1640培养基与脾细胞共培养设为对照组。结果与对照组相比,ELISA和q RT-PCR检测显示在原头蚴为1 000/ml和囊液蛋白质量浓度为2.05 mg/ml时脾细胞产生IL-22增加;流式细胞术检测原头蚴组及囊液组CD4+IL-22+T细胞比例逐渐增加。结论原头蚴及囊液均可促进小鼠脾细胞产生IL-22增加,提示IL-22可能参与宿主防御细粒棘球蚴感染。     

细粒棘球绦虫烯醇酶（EgEnolase）基因的克隆和序列分析

目的细粒棘球绦虫烯醇酶基因（EgEnolase）的克隆和所编码的蛋白质的结构、功能以及应用前景分析。方法利用美国国家生物技术信息中心（NCBI,http：//www.ncbi.nlm.nih.gov/）的在线分析工具BLASTx和瑞士生物信息学研究所的蛋白分析专家系统（ExPaSy,http：//ca.expasy.org/）,以及CBS Prediction Servers提供的蛋白序列在线分析工具,结合Vector NTI suite生物信息学分析软件,从GenBank中细粒棘球绦虫的表达序列标签（EST）数据库中发现烯醇酶的5’端和3’端的EST序列,根据预测的编码区两端序列设计引物,从细粒棘球绦虫青海绵羊分离株中用多聚酶链式反应（PCR）方法扩增其基因组序列,PCR产物克隆到T载体,测序并分析序列中的内含子,以内含子两侧序列的合并序列为引物,采用不对称PCR方法去除其中的内含子序列,预测编码蛋白的结构和功能特征,并分析其应用前景。结果细粒棘球绦虫青海绵羊株烯醇酶基因的基因组序列长为1449bp,含有两个长度分别为78bp和69bp的小内含子。该基因编码433个氨基酸;预测其氨基酸序列中含有一段跨膜区（aa104-124）,N端在膜外,C端在膜内,恰好分为两个不同的功能域,膜内区是执行酶催化功能的主体,Swiss-Model模建的3D结构显示,膜内区由α螺旋和β折叠相间排列形成桶样结构,底物结合位点、催化中心、Mg2＋结合位点等关键位点在空间上紧密靠近,位于桶形结构的中心。该蛋白还有一个潜在的核定位序列aa190-199。该蛋白含有多个T、B细胞表位,且膜外的aa49-57和膜内的aa228-236兼性的T、B细胞表位,线性B细胞表位aa206-213中包含了催化位点Glu210。结论细粒棘球绦虫烯醇酶可能是一个位于虫体皮层表膜具有较好的免疫诊断和疫苗应用前景的膜蛋白,同时还可能进入细胞核调节基因表达。     

Cloning and characterization of Plasmodium vivax thioredoxin peroxidase-1

Reactive oxygen species produced from hemoglobin digestion and the host immune system could have adverse effects on malaria parasites. To protect themselves, malaria parasites are highly dependent on the antioxidant enzymes, including superoxide dismutases and thioredoxin-dependent peroxidases. To date, several thioredoxin peroxidases (TPx) have been characterized in Plasmodium falciparum, but the TPx in Plasmodium vivax has not yet been characterized. The complete sequence of gene coding for thioredoxin peroxidase-1 of P. vivax (PvTPx-1) was amplified by PCR and cloned. Using the recombinant PvTPx-1 (rPvTPx-1), polyclonal antibody was produced in mice for immunolocalization of the enzyme in the parasite. The antioxidant activity of rPvTPx-1 was evaluated by mixed-function oxidation assay. PvTPx-1 has two conserved cysteine residues in the amino acid sequence at the positions 50 and 170 which formed a dimer under a non-reducing condition. Using a thiol mixed-function oxidation assay, the antioxidant activity of rPvTPx-1 was revealed. Indirect immunofluorescence microscopy with the specific antibody indicated that PvTPx-1 was expressed in the cytoplasm of the erythrocytic stage of the parasite in a dots-like pattern. The results suggest that P. vivax uses TPx-1 to reduce and detoxify hydrogen peroxides in order to maintain their redox homeostasis and proliferation in the host body.     

Characterization of the antioxidant enzyme, thioredoxin peroxidase, from the carcinogenic human liver fluke, Opisthorchis viverrini

The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for many years. The mechanisms by which it avoids oxidative damage are unknown. In this study, thioredoxin peroxidase (TPx), a member of the peroxiredoxin superfamily, was cloned from an O. viverrini cDNA library. O. viverrini TPx cDNA encoded a polypeptide of 212 amino acid residues, of molecular mass 23.57kDa. The putative amino acid sequence shared 60-70% identity with TPXs from other helminths and from mammals, and phylogenetic analysis revealed a close relationship between TPxs from O. viverrini and other trematodes. Recombinant O. viverrini TPx was expressed as soluble protein in Escherichia coli. The recombinant protein dimerized, and its antioxidant activity was deduced by observing protection of nicking of supercoiled plasmid DNA by hydroxyl radicals. Antiserum raised against O. viverrini TPx recognized native proteins from egg, metacercaria and adult developmental stages of the liver fluke and excretory-secretory products released by adult O. viverrini. Immunolocalization studies revealed ubiquitous expression of TPx in O. viverrini organs and tissues. TPx was also detected in bile fluid and bile duct epithelial cells surrounding the flukes 2 weeks after infection of hamsters with O. viverrini. In addition, TPx was observed in the secondary (small) bile ducts where flukes cannot reach due to their large size. These results suggested that O. viverrini TPx plays a significant role in protecting the parasite against damage induced by reactive oxygen species from inflammation.     

Echinococcus granulosus calreticulin: molecular characterization and hydatid cyst localization

Hydatidosis, whose etiological agent is the larval stage of the platyhelminth parasite Echinococcus granulosus, is a major zoonotic public health problem and causes great economic losses in many countries, affecting humans and livestock species. Calreticulin (CRT) is a multifunctional and conserved chaperone calcium-binding protein, present in every cell of higher organisms, except erythrocytes. In other parasites species, CRT emerges as a key modulator of several immunological aspects of their relationships with their vertebrate hosts. We report herein the cloning of two DNA segments coding for sequences of E. granulosus CRT (EgCRT). Together with another partial sequence available at the NCBI database, a complete EgCRT cDNA sequence is now proposed. EgCRT mRNA is equally expressed in fertile and infertile hydatid cysts germinal layers, as well as in protoscoleces. In hydatid cysts, EgCRT is a 50 kDa protein, expressed in germinal layer and protoscoleces, mainly in perinuclear and cytoplasmic zones.     

Immunological characterization of Echinococcus granulosus cyclophilin, an allergen reactive with IgE and IgG4 from patients with cystic echinococcosis

By immunological screening of a cDNA library derived from protoscoleces of Echinococcus granulosus with IgE from patients with cystic echinococcosis (CE) and allergic manifestations, we isolated a protein identical to E. granulosus cyclophilin. The protein, named EA21, has close homology with Malassezia furfur cyclophilin allergen (Mal f 6) and with human cyclophilin. Using immunoblotting (IB) with a polyclonal antibody specific to EA21, we identified E. granulosus cyclophilin both in protoscoleces and in sheep hydatid fluid. Of the 58 sera from patients with CE, 29 (50%) were IgE positive to EA21, whereas, despite the high sequence homology, none were IgE positive to Mal f 6 or human cyclophilin. Only 26 of the 58 patients (45%) had IgG specific to EA21, whereas all patients (100%) had IgG specific to Mal f 6 and human cyclophilin. IB analysis showed that serum IgE-binding reactivity to EA21 differed significantly in patients with and without allergic reactions (20 of 25, 80% versus nine of 33, 27%; P < 10(-4)). Conversely, five of the 25 patients who had CE-related allergic manifestations (20%) and 21 of the 33 who did not (63%) had specific IgG4 (P = 10(-3)) and total IgG to EA21. EA21 induced a proliferative response in 15 of 19 (79%) patients' PBMC regardless of the allergic manifestations, but it induced no IL-4 production. Overall, these findings suggest that E. granulosus cyclophilin is a conserved, constitutive, parasite protein that does not cross-react with cyclophilins from other organisms and is involved in the allergic symptoms related to CE.     

细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗诱导的保护力观察

目的探讨细粒棘球绦虫（Eg）转Eg95-EgA31融合基因苜蓿疫苗免疫BALB/c小鼠后对Eg原头节攻击感染的保护性作用。方法热絮凝法提取转基因苜蓿的叶蛋白,配成浓度为20μg/μl。分别用100μl（约含1μg融合抗原）口服灌胃和10μl（约含0.1μg融合抗原）滴鼻接种免疫BALB/c小鼠,每3d免疫1次,连续免疫2月,同时设转空质粒（pBI121）苜蓿叶蛋白及正常苜蓿叶蛋白对照组。末次免疫后8周,用Eg原头节进行攻击感染（腹腔注射50个Eg原头节/每只小鼠）,感染后24周剖杀各组小鼠,检获包囊质量,计算囊重减少率;采眼球血,常规酶联免疫吸附试验法（enzyme-linked immunosorbent assay,ELISA）检测血清中IgG及其亚类和IgE水平。结果疫苗口服灌胃接种组小鼠检获包囊质量明显降低,囊重减少率为64.1%,与正常蛋白对照组相比,差异有统计学意义（P〈0.05）,血清中IgG、IgG2b和IgE水平显著高于正常苜蓿叶蛋白对照组,其值分别为0.175±0.013、0.096±0.028和0.096±0.028。结论细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗口服接种能使免疫鼠获得保护力以抵抗Eg原头节攻击感染,IgG、IgG2b和IgE在疫苗诱导的保护力中起重要作用。     

Molecular cloning of an Echinococcus granulosus protein expressing an immunogenic epitope of antigen 5.

cDNA was synthesized from RNA extracted from Echinococcus granulosus protoscoleces and cloned in the lambda gt11 expression vector. A pool of 5 E. granulosus patient sera was used to screen the library and allowed the selection of 13 clones. Ten of these were shown to be identical, among which clone 6 (Eg6) was chosen for further analysis. The nucleotide sequence (456-bp) presented an entire open reading frame coding for 152 amino acids. The fusion protein (FP6) was recognized by a mouse monoclonal antibody (EG 02 154/12) specific for E. granulosus antigen 5. Moreover, the presence of antibodies to FP6 seemed to be correlated to the ability of sera from hydatidosis patients to immunoprecipitate antigen 5. These results indicate that the cloned protein could be used as a standardized antigen for the diagnosis of hydatidosis.     

Echinococcus granulosus: occurrence of ecdysteroids in protoscoleces and hydatid cyst fluid

Both free ecdysteroids and hydrolysable polar conjugated ecdysteroids were detected in protoscoleces of Echinococcus granulosus from the equine host, and in hydatid cyst fluid from the same source. Comparisons were made of hydatid cyst fluid from E. granulosus infections of three intermediate host species: horses, sheep and humans. Ecdysone and 20-hydroxyecdysone were identified in both protoscoleces and hydatid cyst fluids by high-performance liquid chromatography monitoring fractions by radioimmunoassay, and by capillary gas chromatography/mass spectrometry (selected ion monitoring). The free ecdysteroid fractions of hydatid cyst fluid from horses and sheep also contained several unidentified, chromatographically unique, immunoreactive compounds which were refractory to hydrolysis with a crude Helix pomatia aryl sulphatase enzyme preparation.     

Effect of octenidine dihydrochloride on viability of protoscoleces in hepatic and pulmonary hydatid diseases

BACKGROUND: Use of effective scolicidal agents during puncture, aspiration or injection of a scolicidal agent and reaspiration (PAIR) and surgery for hydatid cysts are essential to reduce the recurrence rate. In this in vitro study, we tried to determine the scolicidal property of a new agent, octenidine dihydrochloride, and of various agents in different concentrations and exposure times. MATERIAL AND METHODS: Echinococcus granulosus protoscoleces were obtained from six patients with liver (n=3) and lung (n=3) hydatid cysts. Various concentrations of octenidine dihydrochloride (0.1%, 0.01% and 0.001% diluted form), povidone iodine (10%, 1% and 0.1% diluted) and 20% saline were used in this study. Viability of protoscoleces was determined with dye-uptake (0.1% eosin) and flame cell activity. RESULTS: Octenidine dihydrochloride 0.1% had strong scolicidal effect in 15 min and octenidine dihydrochloride 0.01% in 30 min. Sixty percent of protoscoleces lost viability at 5 min with octenidine dihydrochloride 0.1%. Viability ratio decreased to 20% at 10 min, and all of them died at 15 min. Povidone iodine 10% and 1% had strong scolicidal effects after 15- and 30 min of exposure, respectively. Saline 20% killed all the protoscoleces in 30-min exposure. CONCLUSION: Because of the rapid and strong scolocidal effectiveness of octenidine dihydrochloride on protoscoleces, it may be used as a scolocidal agent during both perioperative and in the PAIR method.     

Molecular characterization of P-29, a metacestode-specific component of Echinococcus granulosus which is immunologically related to, but distinct from, antigen 5

In this work the characterization of P-29, a novel 29 kDa antigen from Echinococcus granulosus is reported. E. granulosus was identified while looking for parasite antigens distinct from those present in hydatid cyst fluid. A monoclonal antibody (mAb 47H.PS) prepared against protoscolex components revealed that P-29 is localized to the tegument and rostellum of protoscoleces, and to the germinal layer of the cyst, but it is absent in hydatid cyst fluid or adult worm extracts. Several internal fragments of P-29 showed sequence identity to the amino acid sequence encoded by Eg6, a partial gene sequence reported to code for an epitope of antigen 5 (Ag5), one of the major diagnostic antigens of the parasite. We confirmed that Eg6 encodes a sub-fragment of P-29 by mapping the epitope of mAb 47H.PS, and isolating the full length P-29 cDNA. Since Eg6 had been, postulated to encode a fragment of Ag5, we specifically studied the relationship of P-29 and Ag5 by: (i) examining the cross-reactivity displayed by different mAbs; (ii) comparison of their peptide finger prints; and (iii) a comparative study of their diagnostic value. Our results prove unequivocally that P-29 and Ag5 are immunologically related, but different proteins, raising several questions on the current knowledge of Ag5.     

Thioredoxin peroxidase secreted by Fasciola hepatica induces the alternative activation of macrophages

Alternatively activated macrophages (AAMphi) are primarily associated with the chronic stages of parasitic infections and the development of a polarized Th2 response. We have shown that Fasciola hepatica infection of BALB/c mice induces a polarized Th2 response during both the latent and chronic stage of disease. The activation status of macrophages was analyzed in this model of helminth infection by evaluating the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arg1. AAMphi were recruited to the peritoneum of mice within 24 h of F. hepatica infection and after intraperitoneal injection of parasite excretory-secretory (ES) products. Administration of a recombinant antioxidant thioredoxin peroxidase (TPx), which is contained within the ES products, also induced the recruitment of AAMphi to the peritoneum. In vitro studies showed that this recombinant TPx directly converts RAW 264.7 macrophages to an alternatively activated phenotype characterized by the production of high levels of interleukin-10 (IL-10), prostaglandin E(2), corresponding with low levels of IL-12. Our data suggest that the Th2 responses induced by the helminth F. hepatica are mediated through the secretion of molecules, one of which is TPx, that induce the recruitment and alternative activation of macrophages.