微小扇头蜱丝氨酸蛋白酶抑制剂15蛋白生物信息学分析及原核表达

目的了解微小扇头蜱丝氨酸蛋白酶抑制剂15(serine protease inhibitor 15,RmS-15)蛋白的生物学特性,并进行分子克隆和原核表达。方法对RmS-15进行扩增测序,构建系统进化树了解进化关系;采用生物信息学手段分析其理化性质、信号肽、二级和三级结构;利用DNA star和ABCpred工具来预测RmS-15蛋白潜在的B淋巴细胞抗原表位;通过SYF-PEITHI和IEDB预测潜在T淋巴细胞抗原表位;采用大肠杆菌原核表达系统获取微小扇头蜱RmS-15重组蛋白并进行纯化。结果成功扩增出1212 bp的RmS-15基因,可编码403个氨基酸;系统发育树结果显示,RmS-15蛋白在不同蜱虫氨基酸序列中具有较高的保守性,与镰形扇头蜱和血红扇头蜱的亲缘关系最近;理化性质分析结果显示,RmS-15蛋白在N末端中存在20个氨基酸的信号肽,相对分子量大小和理论等电点分别为44000和6.68,平均亲水性为0.001,属于亲水性稳定蛋白;抗原性分析结果表明,RmS-15蛋白B细胞抗原表位优势片段为分别为102~112 aa、147~152 aa和207~215 aa,T细胞抗原表位优势片段为分别为3~11 aa、6~14 aa、27~33 aa和34~37 aa。蛋白表达结果显示,RmS-15蛋白在0.8 mmol/L异丙基硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG),16℃条件下诱导24 h后发现在上清液中有较高的表达量,并且纯化出蛋白分子量为44000、条带单一目的蛋白。结论成功建立微小扇头蜱RmS-15蛋白的原核表达和纯化方法,生物信息学分析提示其有较强的抗原性,具备发展为蜱虫候选疫苗的潜质。     

Immunoglobulin G binding proteins in male Rhipicephalus appendiculatus ticks

Three abundant immunoglobulin G binding proteins (IGBPs) of29kDa (IGBP-MA), 25kDa (IGBP-MB), and 21 kDa (IGBP-MC) were isolated from partly fed male Rhipicephalus appendiculatus ticks using an agar-ose-IgG column. After separating the proteins by SDS-PA GE, each individual IGBP band was cutou tfrom the gel and used to raise antiserum in guinea pigs. Using immuno-blotting, each of the three IGBPs was shown to be antigenically distinct, and specific for feeding male R. appendiculatus ticks. They were detected in the salivary glands of male ticks that had been feeding in either the presence of absence of female ticks, and appeared to be glycoproteins. The smallest protein, IGBP-MC, bound to guinea pig IgG (on which species the ticks were fed) and also bound to human and bovine IgG, whereas IGBP-MA and possibly IGBP-MB only bound to guinea pig IgG. The function of IGBPs in tick salivary glands and their significance in male ticks are discussed.     

Comparative efficacy of rHaa86 and rBm86 against Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus

Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus are the most economically important tick species in India and other tropical and subtropical regions of the world and transmit pathogens causing animal and human diseases. We demonstrated that vaccination of animal by rHaa86 could be used for the control of both H. a. anatolicum and R. (B.) microplus infestations. By comparing the efficacy of rHaa86 and rBm86, it was observed that vaccine based on rHaa86 will be more effective in controlling homologous challenge infestations (68·7% against larvae and 45·8% against adults). The results of this trial demonstrated that species-specific antigens are the better choice for vaccine development and could serve as an effective tool for the integrated control of H. a. anatolicum.     

Genetic analysis of ticks belonging to the Rhipicephalus sanguineus group in Latin America

Phylogenetic analyses based on mitochondrial 16S rDNA sequences were generated from Rhipicephalus sanguineus group specimens collected in 29 localities among 9 Latin-American countries, plus ticks collected in South Africa, Spain, and Italy. Sequences from Latin America generated six different haplotypes (A, B, C, D, E, and F). Phylogenetic analyses generated trees that segregated our tick sequences into two distinct clades: one is represented by haplotypes A–C, and South African R. sanguineus and Rhipicephalus turanicus ticks; the second clade is represented by haplotypes D–F, and European R. sanguineus and R. turanicus ticks. When haplotypes A–F are plotted in the Latin America map according to their geographical coordinates, it is clearly seen that haplotypes D–F are restricted to the southern portion of this continent, whereas haplotypes A–C are distributed in areas between northern Mexico and Brazil (except for the extreme south of this last country, where haplotype E was present). Hence, our phylogenetic analyses separated New World specimens of R. sanguineus into two distinct clades, one represented by tropical and subtropical populations (haplotypes A–C), here designated as the ‘tropical’ species. On the other hand, haplotypes D–F are here designated as the ‘temperate’ species because of their distribution in the southern portion of South America. Until recently, it was assumed that the R. sanguineus group was represented by a single species in the New World, namely R. sanguineus. While the present results coupled with recent studies support the presence of at least two species under the taxon R. sanguineus in the New World, they also show that even in the Old World, the taxon R. sanguineus might be represented by more than one species, since our phylogenetic analysis segregated European and South African R. sanguineus ticks into two distinct clades. The same can be applied for Spanish and South African R. turanicus.     

上海市犬体表媒介蜱寄生情况初步调查

目的初步了解上海市犬体表蜱虫寄生情况。方法2011年3-12月,在上海市18个区(县)采用动物体表采集法捕获犬体表蜱类样本,将蜱虫样本带回实验室,在解剖显微镜下观察成蜱形态,通过形态学鉴定蜱种类。结果在上海市18个区(县)共调查犬1 950只,其中在嘉定、闵行、浦东、松江、黄浦、金山6个区的犬体表共捕获蜱虫328只,经实验室鉴定分为2属2种;宠物犬体表寄生的蜱种为血红扇头蜱和长角血蜱,实验犬和警犬体表寄生的蜱种为血红扇头蜱。结论血红扇头蜱是上海市犬体表寄生的优势蜱种,长角血蜱是新发现的寄生于上海市犬体表的硬蜱蜱种;应加强对媒介蜱的防控。     

气候变化对我国微小扇头蜱分布区的影响

目的 分析影响我国微小扇头蜱地理分布的环境因子及微小扇头蜱在我国的潜在适生区,分析气候变化对我国微小扇头蜱分布的影响.方法 检索我国微小扇头蜱地理分布相关的国内外公开发表文献,提取地理位置信息.结合环境因子,应用ArcGIS 10.7软件和最大熵模型,对微小扇头蜱在我国的适生区分布和影响其分布的主导环境因子进行预测.结...     

我国常见蜱的生物学特性及其传播疾病研究进展

蜱是以吸食动物血液为生的寄生生物,可以传播多种病原体,引起宿主患病。我国蜱种多样,分布广泛,多种蜱具有医学重要性。现就我国常见蜱种的生物学特性及其可传播疾病作一综述。     

Molecular and functional characterization of pigeon (Columba livia) tumor necrosis factor receptor-associated factor 3

Tumor necrosis factor receptor-associated factor 3 (TRAF3) plays a key antiviral role by promoting type I interferon production. We cloned the pigeon TRAF3 gene (PiTRAF3) according to its predicted mRNA sequence to investigate its function. The 1704-bp full-length open reading frame encodes a 567-amino acid protein. One Ring finger, two TRAF-type Zinc fingers, one Coiled coil, and one MATH domain were inferred. RT-PCR showed that PiTRAF3 was expressed in all tissues, with relatively weak expression in the heart and liver. In HEK293T cells, over-expression of wild-type, △Ring, △Zinc finger, and △Coiled coil PiTRAF3, but not a △MATH form, significantly increased IFN-β promoter activity. Zinc finger and Coiled coil domains were essential for NF-κB activation. In chicken HD11 cells, PiTRAF3 increased IFN-β promoter activity and four domains were all contributing. R848 stimulation of pigeon peripheral blood mononuclear cells and splenocytes significantly increased expression of PiTRAF3 and the inflammatory cytokine genes CCL5, IL-8, and IL-10. These data demonstrate TRAF3's innate immune function and improve understanding of its involvement in poultry antiviral defense.     

室内饲养条件下三种硬蜱产卵和孵化特性的比较研究

目的:为了认识微小牛蜱和扇头蜱之间的关系。方法:在室内饲养条件下对微小牛蜱(Boophilusmi-croplus)、镰形扇头蜱指名亚种(Rhipicephalushaemaphysaloideshaemaphysaloides)和猛突血蜱(Haemaphysalismontgomeryi)的产卵和孵化特性进行了比较研究。结果:三种硬蜱在产卵期天数上差异不明显(p>0.05),而在孵化期上有极显著差异(p<0.01);微小牛蜱和镰形扇头蜱间除了吸血后体重(p<0.05)、产卵前期(p<0.01)、孵化期(p<0.01)外,其余参数均差异不明显(p>0.05);猛突血蜱与微小牛蜱间仅在产卵前期和产卵期天数上差异不显著(p>0.05),与镰形扇头蜱间仅在产卵期天数上差异不明显(p>0.05),其余各项参数间都有显著或极显著差异;猛突血蜱的生殖效率和孵化率均明显高于微小牛蜱和镰形扇头蜱(p<0.05)。此外,三种硬蜱不仅日产卵量曲线相似,而且产卵量与吸血后体重之间均存在着极显著的正相关关系(r>0.93,p<0.01)。结论:微小牛蜱和扇头蜱之间有较高的相似度。     

Subolesin: A candidate vaccine antigen for the control of cattle tick infestations in Indian situation

Identification of cross-protective tick vaccine antigens is a challenging area of veterinary research. To address this challenge, a recently identified candidate tick protective antigen, Subolesin (SUB), was targeted in this research. The conservation of subolesin ortholog of Hyalomma anatolicum and Rhipicephalus (Boophilus) microplus across different Indian strains was 98.1–99.4% (within species), while at the amino acid level SUB sequence homology was ≥53.2% (between tick species). Recombinant R. (B.) microplus SUB (rBmSu) was produced in Escherichia coli and characterized. Cross-bred cattle male calves (N = 10) were immunized with three doses of 100 μg each of the rBmSu emulsified in 10% Montanide 888 at monthly intervals on days 0, 30 and 60. The control group was injected with PBS in 10% Montanide 888. For the first tick challenge, calves were infested with larvae of R. (B.) microplus generated from 100 mg eggs 2 weeks after last immunization (day 75). The immunization resulted in 16.3%, 8.0%, 9.4%, and 26.1% reduction in female tick numbers (DT), weight (DW), oviposition (DO) and egg fertility (DF), respectively, when compared to controls. In the subsequent challenge on day 105, DT, DW, DO and DF were reduced by 9.0%, 4.1%, 8.6%, and 24.2%, respectively, when compared to controls. The vaccine efficacy (E) was equal to 44.0% and 37.2% after the first and second challenges, respectively. The results showed a positive correlation between antibody titers for both total IgG and IgG1 and E in the second but not in the first tick challenge. These results suggested the possibility of developing a SUB-based vaccine for control of cattle tick infestations under Indian conditions.