蜱半胱氨酸蛋白酶抑制分子研究进展

蜱是以吸血为生的体外寄生虫,能传播多种病原体。控制蜱的传统方法是使用杀虫剂,但会产生药物残留等问题。为了找到控制蜱及蜱传病的新方法,必须在分子水平上了解蜱的相关生理过程。蜱半胱氨酸蛋白酶抑制分子是一类抗蛋白酶分子,主要作用于半胱氨酸蛋白酶的抑制调控。论文介绍了蜱半胱氨酸蛋白酶抑制分子的结构和分类,以及在蜱的吸血、血液消化、蜱的先天免疫及对宿主免疫调节等生理过程的重要作用,同时也综述了半胱氨酸蛋白酶抑制分子具有抗寄生虫和抗癌的作用,为全面了解蜱半胱氨酸蛋白酶抑制分子提供参考。     

蜱功能基因研究进展

蜱是动物体表的一类常见吸血寄生虫,能充当多种重要疾病的媒介,因此必要采取安全的免疫手段控制蜱害,从分子水平上研究蜱可能成为这种构想的最好突破口。在蜱持续吸血、存储病原等系列独特的生理活动中,相关的功能基因调节发挥着重要作用,了解这些基因的分布、种类和功能,对蜱疫苗的研制和生物制剂的开发将具有重要意义。     

蜱与巴贝斯虫之间互作分子的研究进展

蜱(Ticks)是专性吸血的体外寄生虫,其在吸血过程一方面会造成宿主的皮肤损伤和血液流失,另一方面会引起人畜的蜱传病感染,给畜牧业经济和人类健康带来的影响日益严重。巴贝斯虫(Babesia)是由蜱传播的重要血液原虫之一,会引起人和宿主动物发热、贫血、血尿,甚至死亡,严重危害人类健康,阻碍巴贝斯虫在蜱体内的携带和传播是防范人畜感染的第一道屏障。本文综述了蜱-巴贝斯虫相互作用的分子,包括蜱中肠中的线粒体电压依赖性阴离子选择性通道多肽(VDAC)、半胱氨酸蛋白酶抑制剂(cystatin)等分子,卵巢中的免疫亲和素、卵黄蛋白原受体等分子,以及巴贝斯虫的转甲基酶、HAP2等,以期为后续蜱-巴贝斯虫的互作机制研究提供参考,进一步为蜱和巴贝斯虫疫苗和药物的研发提供信息和基础。     

蜱源蛋白酶及其应用前景

蜱源蛋白酶在蜱和蜱传播疾病的致病机制中发挥重要的生理功能,但对于定性的寄生原虫蛋白鬃酶来说了解得还不很透彻,为了进一步研究蜱源蛋白酶的功能及其特征,筛选出更有价值的蛋白酶基因作为抗蜱疫苗候选抗原基因,从已有的蜱源蛋白酶生理作用角度介绍了蜱源蛋白酶的重要性,提出了将来研究蜱蛋白酶要攻克的难点,为我国采用分子文库的方法筛选抗蜱疫苗候选抗原及研制化学药物提供资料.     

蜱半胱氨酸蛋白酶分子研究进展

蜱作为体表吸血节肢动物,可通过采食宿主血液传播多种病原体,是一种重要媒介。半胱氨酸蛋白酶是一类分布广泛具有一定保守性的蛋白酶,在蜱生理学中发挥重要作用。本文对蜱半胱氨酸蛋白酶的研究进展进行了综述,重点介绍了组织蛋白酶、天冬酰胺内肽酶和半胱天冬氨酸蛋白酶,及参与自噬反应的半胱氨酸蛋白酶的功能及相互作用。     

镰形扇头蜱卵巢中丝氨酸蛋白酶抑制分子群的克隆和生物信息学分析

丝氨酸蛋白酶抑制分子(serine protease inhibitor,Serpin)参与多种生物学过程,对蜱的吸血发育、生殖产卵和先天免疫系统反应等具有重要作用,在蜱体内以多分子形式存在。为了研究蜱生殖相关的Serpin分子群,本研究通过构建镰形扇头蜱(Rhipicephalus haemaphysaloides)卵巢转录组测序数据库,发现并克隆了10条Serpin家族基因(RhS),其中3条为分泌型Serpin,6条为细胞内Serpin和1条亲缘关系较远的Serpin。本研究通过生物信息学方法分析其理化性质、遗传进化关系,预测其结构和功能,为镰形扇头蜱Serpin分子群的功能解析和应用研究提供了重要基础。     

蜱类唾液的采集方法

<正>蜱是一类以吸血为生的体外寄生虫,对人和动物具有直接和间接双重危害,但迄今为止人们尚无安全、高效、环保的防蜱灭蜱措施。以Bm86抗原为基础的抗蜱疫苗的成功研制和商品化激发了蜱学工作者鉴定功能蛋白、筛选候选抗原的热情,且成效明显。蜱唾液中含有大量的功能蛋白,对蜱吸血活动至关重要,其中一些蛋白具有疫苗开发价值,是分离、鉴定蜱唾液功能蛋白的重要来源。由于蜱个体较小,采集比较困难,制约了很多科研人员的研究进程。近两年,笔者在实践     

亚洲璃眼蜱差异表达新基因P18的克隆和鉴定

为了进行抗蜱和蜱传病疫苗的研究,本实验对亚洲璃眼蜱雌蜱吸血前后唾液腺消减文库中获取的一个全长编码基因P18进行了研究。该基因全长519bp,共编码170个氨基酸,分子量为18.36Ku,等电点为4.28。BLAST分析表明,该基因预测的氨基酸与肩突硬蜱、篦子硬蜱唾液腺的抗凝血小肽有30%～40%低度同源性。将该基因亚克隆到pET-32a( )表达载体,转化BL21(DE3)宿主菌,经IPTG诱导,重组融合蛋白以可溶性形式高效表达。将可溶性重组蛋白免疫小鼠后获得的抗血清。经免疫印迹分析表明,该重组蛋白抗体可特异性的识别半饱雌蜱唾液腺中的天然蛋白抗原,而未吸血雌蜱唾液腺中则不显现特异条带。RT-PCR结果进一步证实,该基因在蜱吸血后的唾液腺中差异表达。     

蜱的化学和免疫学防治研究进展

蜱既是常见的吸血外寄生虫,又是人和动物许多重要疾病的传播媒介。大量的蜱叮咬宿主动物可导致宿主消瘦、贫血,同时也使动物皮革的质量降低。作为传播媒介,蜱由于可传播原虫、立克次体、支原体、衣原体、螺旋体、细菌和病毒等,在世界范围带来了很大的经济损失。有很多蜱的防治策略,但均存在不足之处,目前主要以化学和免疫学防治为主。利用化学防治较为普遍,但由于使用不当已造成了蜱的耐药性和环境污染。蜱的免疫学防治主要是开发抗蜱疫苗,接种商品化的抗蜱疫苗被证实是一种可行的防治策略,因此疫苗将在未来对蜱的防治中起举足轻重的作用。     

蜱MicroRNA研究进展

蜱是一类专门寄生于脊椎动物的体表寄生虫,是继蚊子之后的第二大传播媒介。它们传播多种疫病,不仅给畜牧业造成极大的经济损失,而且还引起人类发生疾病甚至死亡。MicroRNA(简称miRNA)是18nt~25nt的非编码小RNA,具有组织特异性和阶段特异性的特点。miRNA作用于靶基因的3′端非翻译区以抑制其表达或者降解mRNA,进而在后转录水平发挥着重要的生物学调控作用,如生长发育和先天性免疫等。有研究显示,miRNA可能调控幼蜱的生长发育,并对蜱虫吸血和血液消化有一定的作用。论文综述了miRNA在蜱体内的研究概况及其生物学功能的研究进展,以期为蜱的研究提供参考。     

Ixodid-host immune interaction. Identification and characterization of relevant antigens and tick-induced host immunosuppression

Ixodid ticks are the most important vectors of pathogens to domestic and wild animals. It is established that cattle and laboratory animal species acquire resistance to tick infestation; acquired resistance has an immunological basis consisting of cell-mediated, antibody-mediated and complement-dependent effector mechanisms. Even though acquired resistance to tick feeding is expressed, host immune competence is possibly impaired during the course of tick feeding. Ixodid-induced transient immunosuppression could possibly facilitate the transmission of vector-borne pathogens and/or enhance tick feeding capabilities in the presence of a host immune response to the hematophagous arthropod. Tick tissue extracts have been used to artificially induce resistance to ixodid feeding, and this has become an area of increasing interest as a possible strategy for tick control. It is essential to have defined antigenic molecules for analysis of host responses to infestation, characterization of immunopathologic processes and for vaccine development. This report focuses on attempts to identify, characterize and isolate tick immunogens. Protein immunoblotting, utilizing sera from animals of different genetic composition and infestation patterns, was used to detect a number of tick polypeptides which are reactive with sera of infested hosts. It is clear that infestation with one ixodid species stimulates antibodies reactive with molecules derived from the sensitizing species and/or tick species in the same genus or different genera. This approach is used to identify molecules that are good candidates for use in immunization studies and for analysis of mechanisms involved in acquisition and expression of resistance to tick feeding.     

Cloning and characterization of a plasminogen-binding enolase from the saliva of the argasid tick Ornithodoros moubata

Significant amounts of enolase have recently been found in the saliva of the argasid tick Ornithodoros moubata, raising the question as to what the function of enolase in the tick–host interface is. Enolase is a multifunctional glycolytic enzyme known to act as a plasminogen receptor on cellular surfaces, promoting fibrinolysis and extracellular matrix degradation. Fibrinolysis could be important for ticks to dissolve clots that might be formed during feeding as well as to prevent clotting of the ingested blood meal in the tick midgut. Additionally, enolase-mediated extracellular matrix degradation could contribute to the tick feeding lesion. Moreover, previous observations suggested an additional antihaemostatic role for O. moubata enolase as a P-selectin antagonist ligand. Accordingly, the aim of the present study was to investigate the potential role of the O. moubata salivary enolase as a plasminogen receptor and P-selectin ligand, and to evaluate its potential as an antigen target for anti-O. moubata vaccines. The study included the cloning, sequencing and recombinant production of the O. moubata enolase, plasminogen binding and activation assays, P-selectin binding assays, animal immunization trials, and RNAi knockdown of the enolase gene. Here we confirmed that enolase is secreted to the saliva of the tick and provide convincing evidence for a role of this salivary enolase as a plasminogen receptor, most likely stimulating host fibrinolysis and maintaining blood fluidity during tick feeding. The RNAi experiments and immunization trials indicated that enolase could be also involved in the regulation of tick reproduction, suggesting new potential control strategies. Finally, the P-selectin binding experiments demonstrated that this enolase is not a P-selectin ligand.     

Tick vaccines and the transmission of tick-borne pathogens

Ticks transmit pathogens that cause diseases which greatly impact both human and animal health. Vaccines developed against Boophilus spp. using Bm86 and Bm95 tick gut antigens demonstrated the feasibility of using vaccines for control of tick infestations. These vaccines also reduced transmission of tick-borne pathogens by decreasing exposure of susceptible hosts to ticks. The recently discovered tick antigens, 64P putative cement protein and subolesin involved in the regulation of tick feeding and reproduction, were also shown to reduce tick infestations. These antigens, together with the TROSPA receptor for Burrelia burgdorferi OspA were effective against tick-borne pathogens by reducing the infection levels in ticks and/or the transmission of the pathogen. Development of a vaccine targeted at both the tick vector and pathogen would contribute greatly to the control of tick infestations and the transmission of tick-borne diseases. These results have demonstrated that tick vaccines can be developed for control tick infestations and show promise for the prevention of the transmission of tick-borne pathogens.     

Anti-chemokine activities of ixodid ticks depend on tick species,developmental stage,and duration of feeding

Ixodid ticks require comparatively large bloodmeals for their development and survival. Blood-feeding elicits signaling events in the host leading to wound healing responses (hemostasis, inflammation, and tissue repair) and immunity. Bioactive molecules present in tick saliva sabotage these host responses at several levels. One of them is neutralization of cellular communication by binding of specific saliva molecules to cytokines that have important roles in innate and adaptive immunity. Chemokines are a subset of cytokines having chemotactic activities. We show anti-chemokine activities in salivary gland extracts (SGE) of adult Rhipicephalus appendiculatus ticks against human chemokines CXCL8, CCL2, CCL3, CCL5, and CCL11. At comparable protein concentrations, male Ixodes ricinus SGE showed activity against all the chemokines; SGE of female I. ricinus had comparatively lower levels of activity against all the chemokines but no detectable activity against CCL5 and CCL11. However, when the equivalent of a single pair of salivary glands was tested, male I. ricinus showed little or no activity against CCL3 and CCL5. No fundamental differences in activity were observed against mouse compared with human chemokines. A comparison with previously published data for Dermacentor reticulatus and Amblyomma variegatum indicates that the level of anti-cytokine activity depends on the species, developmental stage (adult or nymph), and amount of SGE used, as well as on the number of days the tick has been feeding.     

Tick Vaccines and the Transmission of Tick-Borne Pathogens

Ticks transmit pathogens that cause diseases which greatly impact both human and animal health. Vaccines developed against Boophilus spp. using Bm86 and Bm95 tick gut antigens demonstrated the feasibility of using vaccines for control of tick infestations. These vaccines also reduced transmission of tick-borne pathogens by decreasing exposure of susceptible hosts to ticks. The recently discovered tick antigens, 64P putative cement protein and subolesin involved in the regulation of tick feeding and reproduction, were also shown to reduce tick infestations. These antigens, together with the TROSPA receptor for Burrelia burgdorferi OspA were effective against tick-borne pathogens by reducing the infection levels in ticks and/or the transmission of the pathogen. Development of a vaccine targeted at both the tick vector and pathogen would contribute greatly to the control of tick infestations and the transmission of tick-borne diseases. These results have demonstrated that tick vaccines can be developed for control tick infestations and show promise for the prevention of the transmission of tick-borne pathogens.     

Hyalomma anatolicum anatolicum and Hyalomma dromedarii (Acari: Ixodidae) imbibe bovine blood in vitro by utilizing an artificial feeding system

Hard ticks have great importance because of blood feeding and transmitting dangerous human and animal diseases. Each year, they cause a lot of economical damage to the livestock industry. Control of ticks and tick-borne diseases is a major priority in most parts of the world and many studies have been done in this field. We know that studying haemoparasites and assessing the effect of different compounds on ticks requires a lot of money, support and sometimes it is time consuming. Considering all of these problems, today, in some research laboratories throughout the world, artificial in vitro feeding of ticks has become common. Development and application of such methods provide a cheap and accessible background for investigating haemoparasitic diseases under controlled conditions. For the first time we report successful in vitro feeding of two important ixodid ticks of the genus Hyalomma.     

Random sequencing of cDNA library derived from partially-fed adult female Haemaphysalis longicornis salivary gland

A cDNA library was constructed from salivary glands of partially-fed adult female Haemaphysalis longicornis (hard tick). Randomly selected clones were sequenced and a total of 633 sequences were analyzed by bioinformatic programs. The sequences were grouped into 213 clusters, with each cluster being considered to be composed of mRNAs derived from the same gene or closely related genes. About 36% of the mRNA sequences showed significant similarity to known proteins in the non-redundant protein database by the NCBI blastx program and appeared to be coding for functional predicted proteins, whereas the remaining 64% had no similar sequences. Two thirds of the predicted proteins were annotated as basic cellular proteins (housekeeping proteins). Among the functional predicted protein sequences, other than the housekeeping proteins, several protease inhibitors including anticoagulants, two metalloproteases and a potential immunosuppressive protein could be identified. These proteins may play important roles during tick feeding and could be novel anti-tick vaccine candidates.     

Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus

The acquisition and transmission of rickettsial pathogens by different tick developmental stages has important epidemiological implications. The purpose of this study was to determine if male Rhipicephalus sanguineus can experimentally acquire and transmit Ehrlichia canis in the absence of female ticks. Two trials were performed where nymphal and male R. sanguineus were simultaneously acquisition fed on the same infected donor hosts, and transstadially or intrastadially exposed male ticks were fed on separate pathogen-free dogs as a test for transmission. A single-step p30-based PCR assay was used to test canine and tick hosts for E. canis infections before and after tick feeding. E. canis was detected after either intrastadial or transstadial passage in male ticks, the organism remained detectable in both tick groups after transmission feeding, and both tick groups transmitted the rickettsia to susceptible dogs. Infection of dogs via tick feeding resulted in milder clinical signs and lower antibody titers than intravenous inoculation of carrier blood, but further investigation is needed to understand the mechanisms responsible for this observation. These results demonstrate that male R. sanguineus can take multiple feedings, and that they can both acquire and transmit E. canis in the absence of female ticks. This tick development stage could be important in transmission of E. canis, and perhaps related pathogens, between vertebrate hosts under natural and experimental conditions.