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。     

微小牛蜱一个抗菌多肽编码基因的克隆和表达

为了进行抗蜱及蜱传病疫苗的研究,本研究根据微小牛蜱巴西株报道的一种抗菌多肽核苷酸序列设计引物,从微小牛蜱中国安徽株克隆到该抗菌多肽基因,全长383bp,编码110个氨基酸残基,该蛋白预测的分子量为12.2ku,等电点为4.87.经同源性比较,该微小牛蜱巴西株抗菌多肽基因有100%的相同性.经RT-PCR分析表明,该基因在微小牛蜱卵、幼蜱、半饱血雌蜱、饱血雌蜱和雄蜱这几个阶段均有表达.将该基因亚克隆到pET-28a( )表达载体,转化BL21(DE3)宿主菌,经IPTG诱导,可成功表达.重组融合蛋白大小为15ku左右,与预期大小一致.初步体外抗菌试验表明,重组蛋白具有一定的抗菌活性.Western-blot显示,兔抗微小牛蜱唾液抗体能够识别重组表达蛋白.     

镰形扇头蜱丝氨酸蛋白酶SP30基因的克隆及原核表达

根据丝氨酸蛋白酶保守性氨基酸序列设计引物,以镰形扇头蜱半饱血雌蜱cDNA为模板,经PCR扩增得到SP30基因的保守区,通过5’RACE(rapid amplification of cDNA Ends)和3’RACE技术得到全长基因。将该基因连入原核表达载体pGEX-4T-1,经IPTG诱导纯化得到重组蛋白。以镰形扇头蜱卵、幼蜱、若蜱、成蜱各发育阶段的cDNA为模板进行Real-timePCR实验。结果显示,SP30基因全长1194 bp,开放阅读框长900 bp,编码299个氨基酸,预计蛋白分子质量30 kDa。Real-time PCR分析结果表明,该基因在四个不同的发育阶段均有不同程度的表达。     

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。     

边缘革蜱铁蛋白1的原核表达与理化特性分析

为探明蜱源铁蛋白1(FER1)在边缘革蜱铁代谢中的功能,本研究经PCR扩增了边缘革蜱源FER1基因编码区(CDs),测序结果显示FFER1基因CDs区为519 bp;BLAST分析其与硬蜱科常见蜱FER1的氨基酸序列同源性,利用Mega 6.0软件分析了 FER1基因与其他常见蜱该基因之间的系统进化关系;利用Swiss...     

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.     

亚洲璃眼蜱P27基因的克隆和表达模式分析

为了进一步研究蜱吸血时唾液腺的基因表达调控机制,并为蜱疫苗的制备筛选功能性抗原,本试验对从亚洲璃眼蜱雌蜱吸血前后唾液腺消减文库中筛选出的一个具有Poly（A）尾的EST序列P27进行了研究。首先运用RACE技术扩增出该基因的5′端,在此基础上设计引物,扩增出该基因全长,此基因全长827bp,其开放阅读框从第35个碱基始至第706个碱基止,预测分子量为27．28ku,等电点4．22。生物信息学结构分析表明该基因含有跨膜区及多个活性位点,可能与细胞内DNA的转录相关;该基因与现有其他基因同源性很小,为一新基因;RT-PCR结果显示该基因在半饱血雌蜱的唾液腺、蜱壳、中肠均有表达,但在壳中表达丰度稍低。本研究克隆的P27基因序列已登录GenBank,序列号为EU180067。     

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。     

Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction

The potential role of Boophilus microplus as a natural tick vector of Babesia equi and Babesia caballi in Brazilian horses was assessed using nested polymerase chain reaction (PCR)-based marker assay. B. equi merozoite-specific 218bp gene fragment was detected in almost 96% of horse blood samples, and 45.3-62.5% of females, eggs, larvae, and nymphs of B. microplus collected from 47 horses at Campo Grande in the State of Matto Grosso, Brazil. Except for the partially-fed female ticks, the B. caballi-specific 430bp gene fragment was amplified from horse blood samples, and all developmental stages. Parasite DNA from both species was detected in horse blood samples and B. microplus, with the preponderance of B. equi DNA. No DNA samples were positive solely for B. caballi parasite. Only 32% of the Giemsa-stained thin blood smears were positive for Babesia parasites, as against detection of B. equi parasite DNA in 95.7% of the blood samples by nested PCR. We have obtained molecular evidence that strengthens earlier experimental and ultrastructural studies in Brazil incriminating B. microplus as a natural vector of B. equi, and possibly of B. caballi. The detection of B. equi and B. caballi DNA in eggs and larvae of B. microplus is likewise suggestive of the possibility of both transovarial and transstadial parasite transmission in this tick vector.     

Reduced oviposition of Boophilus microplus feeding on sheep vaccinated with vitellin.

The most abundant protein present in Boophilus microplus eggs, vitellin, was isolated and purified as a non-covalent complex of six glyco-polypeptides of Mr 44-107kDa. The protein complex bound haem. Immuno-blots demonstrated that antibodies raised to vitellin recognised a 200kDa polypeptide in the haemolymph of adult female ticks. This is consistent with the general proposal that in arthropods vitellin is derived by proteolytic processing from a large precursor protein, vitellogenin. In parallel with this study, an 80kDa glycoprotein (GP80) was independently purified from larvae of B. microplus using efficacy in vaccination trials as an assay. Antibodies to GP80 also recognised a 200kDa protein in the haemolymph of ticks and a major 87kDa polypeptide present in the vitellin complex. Conversely, antibodies to purified vitellin recognised GP80. The amino-terminal amino acid sequences of the 87kDa vitellin polypeptide and GP80 were identical for at least the first 11 residues and internal peptide sequences from both polypeptides were co-located in a single but incomplete deduced amino sequence of B. microplus vitellogenin. Thus, GP80 is a processed product from vitellogenin and highly related to but not completely identical with the 87kDa vitellin polypeptide. Vaccination trials in the model host sheep were performed with purified vitellin and GP80. Sheep vaccinated with either purified vitellin or GP80 returned significantly reduced numbers of engorged female ticks with decreased weights and reduced oviposition. In contrast, sheep vaccinated with recombinant hexahis-GP80, which was incorrectly folded and not glycosylated showed no significant effects on ticks. It was concluded that vitellin and GP80 could induce immune responses that partially protect sheep from the tick, B. microplus. However, critical protective epitopes are associated with the folding of the protein and/or the oligosaccharides attached to it.     

Molecular analysis of Boophilus spp. (Acari: Ixodidae) tick strains

Boophilus spp. (Acari: Ixodidae) parasitize cattle and other farm and wild animals in tropical and subtropical regions of the world. Ticks belonging to the genus Boophilus have undergone evolutionary processes associated with habitat adaptation following biogeographical separation, resulting in strains with marked morphological differences. We have characterized at the molecular level B. microplus strains from Latin America and Australia, employing sequences derived from the bm86 coding region, an intron located within the bm86 gene, and DNA short tandem repeats (STR). A B. annulatus strain was employed for comparison. The results indicated that variation within the bm86 coding region is higher between B. microplus strains than between some B. microplus strains and B. annulatus. The sequence of the intron was not informative for phylogenetic analysis, varying among individuals of the same strain. Two STRs were identified in B. microplus (STRs BmM1 and BmM2) and one in B. annulatus (STR Ba1). Southern hybridization experiments with STRs BmM1 and BmM2 as a probe revealed the prevalence of dispersed moderately repeated DNA in the genome of B. microplus. The analysis of polymorphism at STR locus BmM1 evidenced differences within and between populations of B. microplus. These results support at the molecular level the existing differences between B. microplus strains and suggest tools to characterize these populations.     

银黑狐内皮素受体B基因（EDNRB）的克隆及其组织表达分析

采用RT-PCR方法对银黑狐内皮素受体B基困（EDNRB）进行了克隆,序列分析表明所克隆的cDNA序列全长为1 636bp,包含整个开放阅读框1 332bp,编码443个氨基酸残基。银黑狐EDNRB基因编码序列与GenBank数据库中的犬、猪、人、牛、兔和小鼠的EDNRB基因核酸序列高度同源,同源性分别为99.0%,90.7%,89.5%,89.1%,86.9%和82.8%。银黑狐EDNRB基因编码氨基酸序列中包含1个信号肽序列和7个跨膜区域,跨膜区氨基酸序列在不同物种中高度保守。EDNRB基因组织表达谱分析表明该基因在银黑狐肺、脑和肝脏组织中表达量较高,而在肌肉、脾和心脏中表达量较低,在胃组织中没检测到该基因的表达。本研究结果为下一步探讨ED-NRB基因核酸序列多态性与银黑狐不同毛色表型的相关性奠定了基础。     

Genetic incompatibility between Boophilus decoloratus (Koch, 1844) and Boophilus microplus (Canestrini, 1888) and hybrid sterility of Australian and South African Boophilus microplus (Acarina: Ixodidae)

Virgin females of both Boophilus decoloratus and Boophilus microplus, when mated with males of the other species, subsequently produced sterile eggs. Counts of spermiophore capsules in female seminal receptacles showed that the males of both species will mate with the females of both species and that B. microplus males show a slightly greater, but statistically insignificant, mating capacity than B. decoloratus males. South African B. microplus females, when mated with an Australian strain of B. microplus males, produced a 62% yield of viable hybrid progeny while the reciprocal cross produced only a 1,82% hatch of non-viable larvae. The hybrids were sterile when interbred and no hatch resulted when the Fl males were backcrossed with parent females. The reciprocal backcross of hybrid Fl females to parent males resulted in a low percentage hatch of non-viable larvae.     

Molecular cloning and sequence analysis of cDNAs encoding for Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus actins

The nucleotide and deduced amino acid sequences of the actins from ticks, Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus, have been determined. Nucleotide sequence analysis showed open reading frames of 1128-nucleotide-long encoding proteins of 376 amino acids with a predicted molecular weight of 41.82 kDa each. Comparison between the nucleic acid and deduced amino acid sequences as well as structural and phylogenetic analyses of these genes confirmed the high similarity among actins from ticks in comparison to other species.     

顿河红豆草凝集素cDNA克隆与序列分析

利用RT-PCR技术从顿河红豆草含浓缩单宁的愈伤组织总RNA中分离克隆了凝集素cDNA.该cNDA包含完整的编码区和3’端非编码区,所编码的氨基酸序列与其它豆科植物凝集素具有较高的同源性。     

Critical evaluation of the modified-adult immersion test with discriminating dose bioassay for Boophilus microplus using American and Australian isolates

Similar adult immersion tests (AITs) for acaricide susceptibility of Boophilus microplus were done in Texas, USA (Mu?oz strain) and in Queensland, Australia (N-strain and Ultimo isolates). Engorged adult female ticks were immersed in one of a series of dilutions of commercial acaricide in water and then incubated at room temperature for 7 days. Data on oviposition were collected 7 days after exposure to acaricide and subjected to probit analysis. For most data, we observed poor fit to the probit model. Substantial differences in both LC50 and LC99 for the susceptible strains occurred between the respective laboratories and confidence intervals for all acaricides and all strains were unacceptably wide. For amitraz, the discriminating concentration (double the LC99.9 or LC99) recommended by FAO was 0.25%, but our estimates ranged from 0.46% to 9000%. For cypermethrin, the recommended DD was 0.0050%, with our estimates ranging from 0.00022% to 0.74%. For coumaphos the recommended DD was 0.50% but our estimates ranged from 0.66% to 130%. Finally, for moxidectin, the recommended DD was 0.10%, while our estimates ranged from <0.0001% to 5.9%. The method does not provide a means to discriminate between amitraz-susceptible and -resistant, nor between cypermethrin-susceptible and -resistant B. microplus.     

Transmission of Babesia spp by the cattle tick (Boophilus microplus) to cattle treated with injectable or pour-on formulations of ivermectin and moxidectin

OBJECTIVE: To assess the efficacy of ivermectin and moxidectin to prevent transmission of Babesia bovis and Babesia bigemina by Boophilus microplus to cattle under conditions of relatively intense experimental challenge. DESIGN: Naive Bos taurus calves were treated with either pour-on or injectable formulations of either ivermectin or moxidectin and then exposed to larvae of B microplus infected with B bovis or larvae or adults of B microplus infected with B bigemina. One calf was used for each combination of haemoparasite, B microplus life stage, drug and application route. PROCEDURE: Groups of calves were treated with the test drugs in either pour-on or injectable formulation and then infested with B microplus larvae infected with B bovis or B bigemina. B bigemina infected adult male ticks grown on an untreated calf were later transferred to a fourth group of animals. Infections were monitored via peripheral blood smears to determine haemoparasite transmission. RESULTS: Cattle treated with either pour-on or injectable formulations of ivermectin and moxidectin became infected with B bovis after infestation with infected larvae. Similarly, larvae infected with B bigemina survived to the nymphal stage to transmit the haemoparasite to animals treated with each drug preparation. Cattle treated with pour-on formulations of ivermectin and moxidectin then infested with adult male ticks infected with B bigemina did not become infected with B bigemina whereas those treated with the injectable formulations of ivermectin and moxidectin did show a parasitaemia. CONCLUSIONS: Injectable or pour-on formulations of ivermectin and moxidectin do not prevent transmission of Babesia to cattle by B microplus. Use of these drugs can therefore not be recommended as a primary means of protecting susceptible cattle from the risk of Babesia infection.