镰形扇头蜱唾液腺cDNA文库的构建

构建了镰形扇头蜱唾液腺 c DNA表达文库。用 Trizol试剂提取镰形扇头蜱唾液腺总 RNA,经反转录合成 c DNA第 1链 ,应用 L D- PCR扩增方法 ,合成双链 c DNA。用 Sfi 内切酶修饰此双链 c DNA,使形成两端分别带有 Sfi A和Sfi B的粘末端。经 CHROMA SPIN- 4 0 0柱纯化 ,收集 4 0 0 bp以上的双链 c DNA片段 ,将其连接于带有 Sfi A和 Sfi B末端的λ Tripl Ex2噬菌体载体 ,经体外包装后 ,以 XL1 - Blue为受体菌构建 c DNA表达文库。经测定 ,库容量为7.0 8× 1 0 5,重组率为 95 .2 %。扩增后的文库滴度为 1 .4 5× 1 0 9pfu/ m L。随机挑取 2 0个克隆转化到质粒 p Tripl Ex2 ,经抽质粒、测序得到 2个细胞色素氧化酶全长基因     

镰形扇头蜱、亚洲璃眼蜱和血红扇头蜱Actin基因的克隆和序列分析

蜱,隶属节肢动物门蛛形纲蜱螨目,寄生于动物体表,以吸食动物血液为生.蜱广泛分布于世界各地,侵袭哺乳动物、爬行动物、鸟类,乃至两栖类等多种动物.蜱和其所携带的病原微生物,如螺旋体、巴贝西虫、贝纳柯克斯体等[1],对动物和人类的健康造成严重危害.肌动蛋白(Actin)是一类进化保守,在多数物种中存在的蛋白质超家族,其基因序列高度同源[2].Actin是细胞组成的基本单位,参与多种生理过程,如细胞运动、胞内运输、细胞质流动、内吞作用等[3].Actin基因及蛋白同时也是多数实验中的首选内参,对完善实验数据、数据分析和实验过程的逻辑性起到重要作用.目前少见对镰形扇头蜱成蜱、亚洲璃眼蜱和血红扇头蜱Actin基因的报道.据此,我们克隆了三种硬蜱的Actin基因,进行了序列分析.     

镰形扇头蜱金属蛋白酶基因全长的克隆和序列分析

本研究从镰形扇头蜱半饱血雌蜱唾液腺cDNA文库的EST序列中筛选了一个含polyA尾的基因序列,经5′-RACE方法得到该基因全长序列。cDNA全长1 566 bp,编码区为23 bp～1 456 bp,编码478个氨基酸残基,该蛋白的预测分子量约为55 Ku,等电点为8.87。RT-PCR分析表明,该基因在镰形扇头蜱未吸血成蜱、半饱血成蜱以及唾液腺、肠中表达。     

镰形扇头蜱肌钙蛋白Ⅱ基因的克隆及其分布

本试验从镰形扇头蜱半饱血雌蜱唾液腺cDNA文库的EST中筛选了1个含polyA尾的基因序列,经5′-RACE方法得到该基因全长序列。经同源性比较,该基因预测的氨基酸序列与长角血蜱肌钙蛋白（GI：14041807）的同源性为84.47%,肌动蛋白结合位点位于147～167氨基酸处,且与长角血蜱肌钙蛋白肌动蛋白结合位点完全相同,表明该基因是镰形扇头蜱肌钙蛋白基因。以镰形扇头蜱基因组DNA为模板扩增到编码肌钙蛋白的基因组序列,序列分析表明该序列不含内含子。RT-PCR分析表明该基因在镰形扇头蜱的卵及其幼蜱、若蜱、成蜱的壳、唾液腺和肠均有表达。     

镰形扇头蜱-新基因的分离、克隆、表达和抗蜱免疫保护作用

目的分离、克隆镰形扇头蜱一新基因。方法本实验从镰形扇头蜱半饱血雌蜱唾液腺cDNA文库的100个EST（Expressed Sequence Tag）序列中选取一个带有polyA尾的序列，经5’RACE（Rapid Amplification of cDNA Ends）得到此序列的全长基因，命名为RhHp2，基因全长1002bp，开放阅读框（ORF）从31bp至879bp，共848bp，编码282个氨基酸。经序列比较，RhHp2基因与其它序列在核苷酸水平上基本无同源性，但在氨基酸水平上与其它物种同源性最高可达50．81％。RhHp2基因连接于PET32a（＋）载体后克隆于表达菌B121，1mM IPTG诱导表达，产生分子量约为55kD的融合重组蛋白，并以不溶性包涵体形式存在。经纯化和体外复性的重组蛋白三次免疫新西兰兔后，将镰形扇头蜱未吸血成蜱和若蜱接种于此免疫兔耳，并对接种48h后的蜱上体数、蜱的饱血数量、饱血时间、饱血重量等进行记录。结果发现蜱的吸血行为受到一定影响，若蜱在48h的减虫率（上体数减少）为58％，在整个吸血过程中的死亡数增加，成蜱的饱血体重显著降低。结论此结果表明RhHp2基因的重组表达产物具有一定的免疫保护作用。     

镰形扇头蜱、亚洲璃眼蜱和血红扇头蜱Aclin基因的克隆和序列分析

蜱,隶属节肢动物门蛛形纲蜱螨目,寄生于动物体表,以吸食动物血液为生。蜱广泛分布于世界各地,侵袭哺乳动物、爬行动物、鸟类,乃至两栖类等多种动物。蜱和其所携带的病原微生物,如螺旋体、巴贝西虫、贝纳柯克斯体等,对动物和人类的健康造成严重危害。     

镰形扇头蜱重组肌钙蛋白I抗蜱免疫保护效果的初步分析

为了评估镰形扇头蜱肌钙蛋白I（TroponinI,TnI）作为候选疫苗分子的潜力,应用肌钙蛋白I重组蛋白进行了抗蜱免疫实验。将重组蛋白按常规方法免疫新西兰兔,三次免疫后分别用若蜱、成蜱进行攻虫实验,观察其免疫保护效果。结果发现蜱的吸血行为受到一定的影响,与对照相比,若蜱和成蜱吸附率、饱血率、饱血体重和死亡率有显著差异,而在饱血时间上差异不显著,说明TnI基因的重组表达产物具有一定的免疫保护作用。     

镰形扇头蜱微管蛋白基因的分离和cDNA序列分析

分离和鉴定镰形扇头蜱（Rhipicephalus haemaphysaloides,Rh）新基因,为防治镰形扇头蜱提供药物靶标或候选疫苗。从镰形扇头蜱唾液腺抑制消减杂交文库中筛选了一条编码微管蛋白的基因片段,通过3＇-RACE的方法得到全长序列;采用生物信息学技术等对该cDNA进行分析。获得1个镰形扇头蜱新基因-微管蛋白基因（RhM1）,全长680bp,开放阅读框（ORF）全长537bp,编码179个氨基酸。编码蛋白的理论分子量为20KDa,等电点为4.71;抗原表位可能位于cDNA序列N末端第18~25氨基酸处。     

镰形扇头蜱肌钙蛋白Ⅰ基因的克隆及其在大肠杆菌中的表达

目的克隆镰形扇头蜱肌钙蛋白Ⅰ(TnI)的cDNA并进行表达及鉴定。方法用RT-PCR方法从镰形扇头蜱总RNA中克隆编码肌钙蛋白Ⅰ的cDNA片段,将该cDNA连接到pET-32a( )表达载体,并转入宿主菌BL21(DE3),经IPTG诱导表达,并对表达产物进行Western blot分析。结果获得镰形扇头蜱的eDNA,并在大肠杆菌中以包含体的形式高效表达,Westem blot分析表明,抗TnI重组蛋白兔血清可以识别蜱体内的TnI,并在22 kDa和36 kDa之间有两条带。结论蜱体内可能同时存在两个大小不等的TnI,为进一步的研究打下基础。     

Comparative infection rates of Theileria parva lawrencei in salivary glands of Rhipicephalus appendiculatus and Rhipicephalus zambeziensis

Three cattle, which had been experimentally infected with Theileria parva lawrencei and maintained as carriers of the infection, were each infested simultaneously with clean nymphal Rhipicephalus appendiculatus and Rhipicephalus zambeziensis in ear bags on separate ears. After moulting, the ensuing adult ticks were fed on rabbits for 4 days and their salivary glands were examined for infective stages of the parasite. Microscopic examination revealed significantly higher infection rates in the salivary glands of R. zambeziensis than in R. appendiculatus which may indicate an increased vector efficiency of R. zambeziensis for T. p. lawrencei.     

Identification of Theileria infections in the salivary glands of Hyalomma anatolicum anatoli cum and Rhipicephalus appendiculatus using isoenzyme electrophoresis

Lysates prepared from the salivary glands of uninfected Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus and from ticks of these species infected with 2 strains of Theileria annulata and 1 strain of T. parva respectively have been examined for enzyme polymorphism using thin layer starch gel electrophoresis. Representative enzymes from 3 of the major metabolic pathways, the tricarboxylic acid cycle, the glycolytic pathway and the pentose phosphate pathway were investigated. Only 1 enzyme, glucose phosphate isomerase, showed differences between the host cell activity and the parasite activity and also inter- and intraspecific differences between the parasites. This technique represents a useful complement to the present histochemical methods for identification of Theileria infections in the salivary glands of ticks.     

镰形扇头蜱组胺结合蛋白全长基因的克隆与分析

从本实验室构建的镰形扇头蜱（Rhipicephalus haemaphysaloides）抑制消减杂交cDNA文库中选择了1条可能编码组胺结合蛋白（HBP）的EST序列（RhHBP）,以5′末端快速扩增的方法（5′RACE）扩增获得5′末端序列,拼接获得全长基因。DNAMAN（v4.0）、DNAstar（v4.0）和Genetyx（v4.0）软件分析阅读框、编码产物、序列同源性和系统进化,同时也分析了该基因在吸血前后雌雄蜱体内的表达情况。5′RACE法获得1条约400 bp的DNA片段,克隆测序,拼接获得1条长803 bp的全长基因,编码219个氨基酸残基。该基因在吸血雌蜱唾液腺内特异表达,初步分析该基因是组胺结合蛋白基因。     

镰形扇头蜱P0基因的克隆表达及初步鉴定

P0分子是一种核糖体蛋白,具有抗蜱免疫作用。本文采用RACE技术从镰形扇头蜱(Rhipicephalus haemaphysaloides)中获得了P0全长基因。通过对P0全长基因序列进行分析发现,P0全长基因为1 118 bp,具有一个编码319个氨基酸的开放阅读框,没有信号肽序列。推测P0的蛋白大小约为35 ku。P0与其他蜱种具有很高的同源性,与微小扇头蜱(Rhipicephalus microplus)和长角血蜱(Haemaphysalis longicornis)的P0基因同源性分别为94%和87%。编码区克隆到表达载体p GEX-4T-1中,转化表达宿主菌BL21,可表达出分子量约为60 ku的重组融合蛋白。表达的蛋白以包涵体的形式存在于菌体中。表达产物经GST树脂纯化后可得到纯度较高的目的蛋白。抗P0重组蛋白鼠血清可以识别蜱体内的P0蛋白,大小在35 ku。研究结果将为镰形扇头蜱新型防治技术研究提供基础。     

镰形扇头蜱肌钙蛋白I基因的克隆及其分布

本试验从镰形扇头蜱半饱血雌蜱唾液腺cDNA文库的EST中筛选了1个含polyA尾的基因序列,经5LRACE方法得到该基因全长序列.经同源性比较,该基因预测的氨基酸序列与长角血蜱肌钙蛋白I(GI14041807)的同源性为84.47%,肌动蛋白结合位点位于147-167氨基酸处,且与长角血蜱肌钙蛋白I肌动蛋白结合位点完全相同,表明该基因是镰形扇头蜱肌钙蛋白I基因.以镰形扇头蜱基因组DNA为模板扩增到编码肌钙蛋白I的基因组序列.序列分析表明该序列不含内含子.RT-PCR分析表明该基因在镰形扇头蜱的卵及其幼蜱、若蜱、成蜱的壳、唾液腺和肠均有表达.     

镰形扇头蜱组胺结合蛋白RhHBP基因的克隆和重组表达

为了进行抗蜱及蜱传病疫苗的研究,本试验根据GenBank中的镰形扇头蜱组胺结合蛋白(Rhipicephalus haemaphysaloideshistamine bindingprotein,RhHBP)基因cDNA序列,设计一对特异性引物(内含EcoRI/XhoI酶切位点),经RT-PCR扩增了镰形扇头蜱组胺结合蛋白RhHBP的cD-NA,大小为603 bp,将其克隆到pGEM-Teasy中并测序。在去除编码RhHBP信号肽的核苷酸序列后,将其亚克隆到pGEX-4T-1中,构建重组质粒pGEX-4T-1-RhHBP,将该重组质粒转化大肠杆菌BL21,经终浓度为1 mmol/L IPTG诱导其表达,表达的融合蛋白大小为49 ku,以包涵体的形式存在。Western Blot分析表明,该蛋白具有良好的免疫原性。     

Identification of Rickettsia felis in fleas but not ticks on stray cats and dogs and the evidence of Rickettsia rhipicephali only in adult stage of Rhipicephalus sanguineus and Rhipicephalus haemaphysaloides

Rickettsia spp. are zoonotic pathogens and mainly transmitted by various arthropod vectors, such as fleas, ticks, and lice. Previous epidemiological studies indicated that ectoparasites infested on dogs or cats may be infected by Rickettsia spp., and transmit them to human beings accidentally. In this study, the prevalence of Rickettsia infection was evaluated using fleas and ticks from stray dogs and cats in Taiwan. A total of 158 pools made by 451 cat fleas (Ctenocephalides felis) from 37 dogs and 4 cats were used for analysis. Besides, 386 Rhipicephalus ticks collected from the other 62 stray dogs were included in this study. Nymphal and adult ticks were individually analyzed but larvae were separated into 21 pools for molecular detection. Partial sequencing analysis of the gltA gene was applied for Rickettsia identification. The results showed that 44.3% (70/158) of the cat flea pools were harboring Rickettsia DNA. Although 6.9% (13/187) of adult ticks were infected with Rickettsia, neither larval pools nor nymphal ticks were found to contain Rickettsia DNA. According to the results of sequencing analyses, all Rickettsia PCR-positive cat flea pools were infected with R. felis, and all Rickettsia PCR-positive adult ticks were infected with R. rhipicephali. The results of this study demonstrated that C. felis but not Rhipicephlus sanguineus (the brown dog tick) and Rh. haemaphysaloides collected from stray animals in Taiwan could be infected the zoonotic pathogen R. felis. Moreover, R. rhipicephali was only identified in adult stage of Rhipicephalus sanguineus and Rh. haemaphysaloides.