长角血蜱MLP、BBD基因的获得及部分功能特性研究
摘要
蜱作为重要的病原传播媒介，能够通过吸血性寄生传播细菌、病毒、原虫等多种病原。对我国畜牧业造成了巨大的经济损失，同时也严重威胁着人类的健康。化学药物防控一直以来都是蜱虫防治的主要手段，但是随着化药杀虫剂所带来的副作用不断凸显，使得研发新的防控制剂变成为必然。本研究以在我国分布最广、危害最为严重的长角血蜱（Haemaphysalis longicornis）为研究对象，采用免疫文库筛选及cDNA末端快速扩增（RACE）等技术，克隆、表达出目的基因，并进一步研究了其部分生物学功能，取得了如下成果。
对长角血蜱饥饿雌蜱cDNA表达文库中筛选到的未知基因进行5’RACE扩增，得到948bp的长角血蜱肌肉LIM蛋白（muscle LIM proteins，MLP）的cDNA全长序列。完整的开放式阅读框（ORF）含有321个碱基，编码106个氨基酸，预测蛋白分子大小约为11.3ku，等电点为8.58。设计通用引物采用PCR方法分别从青海血蜱、麻点璃眼蜱、森林革蜱、微小牛蜱及小亚璃眼蜱中扩增获得了MLP基因完整的开放阅读框，并将序列提交到GenBank数据库中，获得了登录号。将其克隆到表达载体pGEX-4T-1中，进行原核表达，分别用SDS-PAGE和Western-blot分析目的蛋白的表达情况和反应原性。结果表明，成功获得了长角血蜱等多个蜱种的MLP基因全长序列，体外高效诱导表达了约39.2ku的不同蜱种的MLP重组蛋白。Western-blot分析表明长角血蜱该重组蛋白具有很强的反应原性，且不同蜱的重组蛋白间具有很强的交叉反应性。用Real-timePCR进行相对定量分析，MLP在长角血蜱卵、幼蜱、若蜱、饥饿成蜱和半饱血成蜱中的表达丰度逐步增高，说明MLP的表达随着蜱的发育及吸血过程的发展表达量明显上调；该基因在半饱血雌蜱表皮中的表达丰度显著高于中肠、卵巢和唾液腺等组织；长角血蜱的卵受低温刺激后MLP的表达量明显下调，这些结果表明该基因可能与蜱的肌肉发育调控相关。动物免疫试验及RNAi实验结果显示该蛋白未表现出明显的抗蜱免疫保护作用。
根据已知物种的同源基因序列设计兼并引物，采用同源扩增的方法，通过3’RACE及5’RACE技术扩增得到了长为1780bp的长角血蜱酮戊二酸双加氧酶（gamma-butyrobetaine,2-oxoglutarate dioxygenase，BBD）基因的全长序列。该基因含有一个1353bp的ORF，编码450个氨基酸，预测的分子量为45.0ku。生物信息学分析表明，该序列含有酮戊二酸双加氧酶典型的保守功能域。设计引物从青海血蜱及森林革蜱中扩增得到了该基因，将序列提交到GeneBank，分别获得了序列号。对长角血蜱BBD基因进行了原核表达，Western blot分析结果表明该重组蛋白具有很好的反应原性，Real-time PCR分析结果显示该基因在长角血蜱不同组织中均有分布，且在中肠中表达水平低于其它组织。在若蜱阶段表达量显著高于卵、幼蜱和成蜱阶段。吸血过程对于该基因表达丰度的影响不明显。RNAi结果显示沉默该基因后雌性成蜱的饱血体重与对照组相比明显下降，同时蜱的产卵过程发生障碍。表明该基因在蜱的能量代谢过程中发挥着重要的作用，可能是通过影响蜱体内的能量代谢过程从而影响蜱的吸血和产卵等生理功能。
Many pathogens such as bacteria, virus and protozotan can transmit by the hard ticks, which seriouslythreat the development of husbandry and the health of mankind. Currently, the most popular tick controlmethod is the application of acaricides. It is necessary to develop an alternative agent of tick control asthe growing number of disadvantages caused by acaricides. The study cloned and expressed the targetgene from Haemaphysalis longicornis, the most important hard tick in China, by the method of rapidamplification of the cDNA ends (RACE) and immunoscreening. To analyzed the biological function andassessed the potential for controlling ticks.
The complete sequence of muscle LIM proteins (MLP) from Haemaphysalis longicornis, which wasscreened from a cDNA library constructed from partial-fed female ticks, was obtained by rapidamplification of the cDNA ends (RACE). The full-length of HLMLP was948bp. The HLMLP cDNAcontains321bp that encode106amino acids with a predicted theoretical isoelectric point (pI) of8.58and molecular weight of11.3ku. The nucleotide sequence of MLP and the deduced amino acid wereanalyzed by the bioinformatics software, and the complete open reading frames were amplified by PCRusing universal primer from ticks of Haemaphysalis qinghaiensis, Hyalomma anatolicum, Hyalommarufipes, Boophilus (Rhipicephalus) microplus, Demacentor silvarum respectively, the PCR product wasinserted into expression vector pGEX-4T-1to construct recombinant plasmid, and the recombinantplasmid was transformed into Escherichia coli BL21(DE3) for expression. The expression andreactogenicity of recombinant protein were analyzed by SDS-PAGE and Western-blot. The expressedfusion protein was approximately39.2ku in molecular weight. Western blot analysis showed that rabbitantiserum against the adults of H. longicornis could recognize the rHLMLP and the protein of differentticks. Real-time PCR analysis showed that this gene expressed in integument was significantly higherthan in other tested tissues of partially fed females, and the HLMLP gene expression is developmentallyregulated throughout the life cycle. Furthermore, the transcripts of the HLMLP mRNA weresignificantly downexpression in response to low temperature stimulation. The result of vaccination withrHLMLP and RNAi showed that there were no apparent obvious anti-tick effections.
The full-length cDNA encoding gamma-butyrobetaine,2-oxoglutarate dioxygenase (HLBBD) wasobtained from H. longicornis by5′RACE and3′RACE. The full-length of HLBBD was1780bp. Thecomplete open reading frame (ORF) contains1353bp and coding for450amino acids with a predictedmolecular weight of45.0ku. The gene included functional domain of gamma-butyrobetaine,2-oxoglutarate dioxygenase. The predicted3D structure of HLBBD based on the structure of humangamma-butyrobetaine,2-oxoglutarate dioxygenase was constructed by Swiss-Model. Western Blotsanalysis showed that the expression product of HLBBD showed the strong reactogenicity. Real-timePCR analysis showed that the expressions of HLBBD were no obvious tissue specificity, and the genewas expressed in larval was lower than other different developmental stages. Additionally, thetranscripts of the gene were not significantly induced by blood feeding. RNAi experiment confirmed that the gene could significantly affect the engorgement weight and mortality in blood feeding, and theexceptional oviposition behavior was obeserved in treated groups. These results confirmed that thisprotein play essential role in the progress of tick feeding and production.
对长角血蜱饥饿雌蜱cDNA表达文库中筛选到的未知基因进行5’RACE扩增，得到948bp的长角血蜱肌肉LIM蛋白（muscle LIM proteins，MLP）的cDNA全长序列。完整的开放式阅读框（ORF）含有321个碱基，编码106个氨基酸，预测蛋白分子大小约为11.3ku，等电点为8.58。设计通用引物采用PCR方法分别从青海血蜱、麻点璃眼蜱、森林革蜱、微小牛蜱及小亚璃眼蜱中扩增获得了MLP基因完整的开放阅读框，并将序列提交到GenBank数据库中，获得了登录号。将其克隆到表达载体pGEX-4T-1中，进行原核表达，分别用SDS-PAGE和Western-blot分析目的蛋白的表达情况和反应原性。结果表明，成功获得了长角血蜱等多个蜱种的MLP基因全长序列，体外高效诱导表达了约39.2ku的不同蜱种的MLP重组蛋白。Western-blot分析表明长角血蜱该重组蛋白具有很强的反应原性，且不同蜱的重组蛋白间具有很强的交叉反应性。用Real-timePCR进行相对定量分析，MLP在长角血蜱卵、幼蜱、若蜱、饥饿成蜱和半饱血成蜱中的表达丰度逐步增高，说明MLP的表达随着蜱的发育及吸血过程的发展表达量明显上调；该基因在半饱血雌蜱表皮中的表达丰度显著高于中肠、卵巢和唾液腺等组织；长角血蜱的卵受低温刺激后MLP的表达量明显下调，这些结果表明该基因可能与蜱的肌肉发育调控相关。动物免疫试验及RNAi实验结果显示该蛋白未表现出明显的抗蜱免疫保护作用。
根据已知物种的同源基因序列设计兼并引物，采用同源扩增的方法，通过3’RACE及5’RACE技术扩增得到了长为1780bp的长角血蜱酮戊二酸双加氧酶（gamma-butyrobetaine,2-oxoglutarate dioxygenase，BBD）基因的全长序列。该基因含有一个1353bp的ORF，编码450个氨基酸，预测的分子量为45.0ku。生物信息学分析表明，该序列含有酮戊二酸双加氧酶典型的保守功能域。设计引物从青海血蜱及森林革蜱中扩增得到了该基因，将序列提交到GeneBank，分别获得了序列号。对长角血蜱BBD基因进行了原核表达，Western blot分析结果表明该重组蛋白具有很好的反应原性，Real-time PCR分析结果显示该基因在长角血蜱不同组织中均有分布，且在中肠中表达水平低于其它组织。在若蜱阶段表达量显著高于卵、幼蜱和成蜱阶段。吸血过程对于该基因表达丰度的影响不明显。RNAi结果显示沉默该基因后雌性成蜱的饱血体重与对照组相比明显下降，同时蜱的产卵过程发生障碍。表明该基因在蜱的能量代谢过程中发挥着重要的作用，可能是通过影响蜱体内的能量代谢过程从而影响蜱的吸血和产卵等生理功能。
Many pathogens such as bacteria, virus and protozotan can transmit by the hard ticks, which seriouslythreat the development of husbandry and the health of mankind. Currently, the most popular tick controlmethod is the application of acaricides. It is necessary to develop an alternative agent of tick control asthe growing number of disadvantages caused by acaricides. The study cloned and expressed the targetgene from Haemaphysalis longicornis, the most important hard tick in China, by the method of rapidamplification of the cDNA ends (RACE) and immunoscreening. To analyzed the biological function andassessed the potential for controlling ticks.
The complete sequence of muscle LIM proteins (MLP) from Haemaphysalis longicornis, which wasscreened from a cDNA library constructed from partial-fed female ticks, was obtained by rapidamplification of the cDNA ends (RACE). The full-length of HLMLP was948bp. The HLMLP cDNAcontains321bp that encode106amino acids with a predicted theoretical isoelectric point (pI) of8.58and molecular weight of11.3ku. The nucleotide sequence of MLP and the deduced amino acid wereanalyzed by the bioinformatics software, and the complete open reading frames were amplified by PCRusing universal primer from ticks of Haemaphysalis qinghaiensis, Hyalomma anatolicum, Hyalommarufipes, Boophilus (Rhipicephalus) microplus, Demacentor silvarum respectively, the PCR product wasinserted into expression vector pGEX-4T-1to construct recombinant plasmid, and the recombinantplasmid was transformed into Escherichia coli BL21(DE3) for expression. The expression andreactogenicity of recombinant protein were analyzed by SDS-PAGE and Western-blot. The expressedfusion protein was approximately39.2ku in molecular weight. Western blot analysis showed that rabbitantiserum against the adults of H. longicornis could recognize the rHLMLP and the protein of differentticks. Real-time PCR analysis showed that this gene expressed in integument was significantly higherthan in other tested tissues of partially fed females, and the HLMLP gene expression is developmentallyregulated throughout the life cycle. Furthermore, the transcripts of the HLMLP mRNA weresignificantly downexpression in response to low temperature stimulation. The result of vaccination withrHLMLP and RNAi showed that there were no apparent obvious anti-tick effections.
The full-length cDNA encoding gamma-butyrobetaine,2-oxoglutarate dioxygenase (HLBBD) wasobtained from H. longicornis by5′RACE and3′RACE. The full-length of HLBBD was1780bp. Thecomplete open reading frame (ORF) contains1353bp and coding for450amino acids with a predictedmolecular weight of45.0ku. The gene included functional domain of gamma-butyrobetaine,2-oxoglutarate dioxygenase. The predicted3D structure of HLBBD based on the structure of humangamma-butyrobetaine,2-oxoglutarate dioxygenase was constructed by Swiss-Model. Western Blotsanalysis showed that the expression product of HLBBD showed the strong reactogenicity. Real-timePCR analysis showed that the expressions of HLBBD were no obvious tissue specificity, and the genewas expressed in larval was lower than other different developmental stages. Additionally, thetranscripts of the gene were not significantly induced by blood feeding. RNAi experiment confirmed that the gene could significantly affect the engorgement weight and mortality in blood feeding, and theexceptional oviposition behavior was obeserved in treated groups. These results confirmed that thisprotein play essential role in the progress of tick feeding and production.
引文
1.白启，刘光远，韩根凤，等.甘肃张家川牛卵形巴贝斯虫的分离及补充传播试验[J].中国兽医科技，1994，9(24)：9-10.
2.柴慧萍，刘光远，张林，等.长角血蜱饥饿雌蜱cDNA表达文库的构建及免疫学筛选[J].中国兽医寄生虫学与寄生虫病杂志，2009，27(1)：2-10.
3.邓国藩，姜在阶.中国经济昆虫志，第39册，蜱螨亚纲硬蜱科.北京：科学出版社，1991：34-43.
4.汪明.兽医寄生虫学[M].北京：中国农业出版社，2003，75-78.
5. Alekseev AN, Chunikhin SP, Rukhkyan MY, et al. Possible role of Ixodidae salivary glandsubstrate s an adjuvant enhancing arbovirus transmission[J]. Meditsinskaya ParazitologiyaiParazitarnye Bolezni,1991,1(5):28–31.
6. Aljamali MN, Sauer JR and Essenberg RCl. RNA interference: applicability in tick research[J].Expppl Acarol,2002,28(1-4):89–96.
7. Aljamali MN, Bior AD,Sauer JR et al. RNA interference in ticks: a study using histamine bindingprotein dsRNA in the female tick Amblyomma americanum[J]. Insect Mol Biol,2003,12(3):299–305
8. Almazan C, Kocan KM, Blouin EF et al. Vaccination with recombinant tick antigens for thecontrol of Ixodes scapularis adult infestations[J]. Vaccine,23(46-47):5294–5298.
9. Almazan C, Machado UB, Kocan KM et al. Characterization of three Ixodes scapularis cDNAsprotective against tick infestations[J]. Vaccine,2005,23(35):4403–4416.
10. Anderson JF and Valenzuela JG. Tick saliva: from pharmacology and biochemistry totranscriptome analysis and functional genomes. In Ticks: Biology, Disease and Control,2008, pp.92–107.
11. Arber S, Halder G, Caroni P. Muscle LIM protein, a novel essential regulator of myogenesis,promotes myogenic differentiation[J]. Cell,1994,79(2):221231.
12. Bach I. The LIM domain: regulation by association[J]. Mech Dev,2000,91(1-2):517.
13. Bieber LL and Monroe RE.The relation of carnitine to the formation of phosphatidyl-beta-ethylcholine by Tenebrio molitor L. larvae[J]. Lipids,1969,4(4):293-8.
14. Boldbaatar D,Sikasunge CS,Battsetseg B et al. Molecular cloning and functional characterizationof an aspartic protease from the hard tick Haemaphysalis longicornis[J]. Insect Biochem MolBiol,2006,36(1):25–36
15. Bowman AS, Dillwith JW, Sauer JR. Tick Salivary prostaglandins: presence, origin andsignificance[J]. Parasitol Today,1996,12(10):388-396.
16. Bremer J. Trends in Biochem[J].Science,1997,(2):207
17. Burd CG and Dreyfuss G. Conserved structures and diversity of functions of RNA-bindingproteins[J]. Science,1994,265(5172):615621.
18. Campos E, Facanha AR, Coata EP, et al. A Mitochondrial Membrane Exopolyphosphatase IsModulated by and Plays a Role in the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus)microplus Embryos[J]. Int J Mol Sci.2011,12(6):3525-3535.
19. Cerutti, H. RNA interference: traveling in the cell and gaining functions?[J]. Trends Genet,2003,19(1):39–46.
20. Clark KA, Bland JM, Beckerle MC. The Drosophila muscle LIM protein, Mlp84B,cooperates withD-titin to maintain muscle structural integrity[J]. Journal of Cell Science,2007,120(1):2066-2077.
21. Crawford A W, Pino JD, Beckerle MC. Biochemical and molecular characterization of the chickencysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with theactin cytoskeleton[J]. J Cell Biol,1994,124(1-2):117127.
22. de la Fuente, Almazan C,Blouin EF et al. RNA interference screening in ticks for identification ofprotective antigens[J]. Parasitol Res,2005,96(3):137–141.
23. de la Fuente, Almazan C, Blouin EF et al. Reduction of tick infections with Anaplasma marginaleand A. phagocytophilum by targeting the tick protective antigen subolesin[J]. Parasitol Res,2006,100(1):85–91.
24. de la Fuente, Almazan C, Machado UB et al.The tick protective antigen,4D8, is a conservedprotein involved in modulation of tick blood ingestion and reproduction[J]. Vaccine,2006,24(19):4082–4095.
25. de la Fuente, Almazan C, Naranjo V et al. Synergistic effect of silencing the expression of tickprotective antigens4D8and Rs86in Rhipicephalus sanguineus by RNA interference[J]. ParasitolRes,2006,99(2):108–113.
26. de la Fuente, Almazan C, Naranjo V et al. Autocidal control of ticks by silencing of a single geneby RNA interference[J]. Biochem Biophys Res Commun,2006,344(1):332–338.
27. de la Fuente and Kocan KM. Strategies for development of vaccines for control of Ixodid tickspecies[J]. Parasite Immunol,2006,28(7):275–283.
28. Demma LJ, Traeger MS, Nicholson WL, et al. Rocky mountains spotted fever from an unexpectedtick vector in Arizona [J]. N Engl J Med,2005,353(6):587-594.
29. Dong Y and Friedrich M. Nymphal RNAi: systemic RNAimediated gene knockdown in juvenilegrasshopper[J]. BMC Biotechnol,2005,1472:5-25
30. Editorial. MLP: A stress sensor goes nuclear[J].Journal of Molecular and Cellular Cardiology,2009,47:423–425.
31. Faisal A and Bughdad I.Ultrastructural study of muscles fibers in Tick Hyalomma (Hyalomma)anatolicum anatolicm(Ixodoidea:Ixodidae)[J]. Pakistan Journal of Biological Sciences,2010,13(17):828-834.
32. Feinberg EH and Hunter CP. Transport of dsRNA into cells by the transmembrane protein SID-1[J].Science,2003,301(5639):1545–1547.
33. Fereira C A, Barbosa M C, Silveira T C, et al. cDNA cloning, expression and characterization ofBoophilus microplus paramyosin[J]. Parasitology,2002,125:265–274.
34. Frederic MV and Ronald JA.Carnitine biosynthesis in mammals Biochem[J],2002,361:417-429.
35. Gao JL, Luo JX, Fan R, et al. Molecular characterization of amyosin alkali light chain-like protein,a “concealed” antigen from the hard tick Haemaphysalis qinghaiensis[J].Vet Parasitol,2007,147(1-2):140–149.
36. Gao JL, Luo JX, Fan R, et al. Cloning and characterization of a cDNA clone encoding troponin Tfrom tick Haemaphysalis qinghaiensis (Acari: Ixodidae)[J]. Comp Biochem Physiol B BiochemMol Bio.2008, l151(3):323-329.
37. Garcia S,Billecocq,Crance JM, et al. Nairovirus RNA sequences expressed by a Semliki Forestvirus replicon induce RNA interference in tick cells[J]. J Virol.2005,79(14):8942–8947.
38. Garcia S, Billecocq,Crance JM, et al.Viral suppressors of RNA interference impair RNA silencinginduced by a Semliki Forest virus replicon in tick cells[J]. J Gen Virol.2006,87(7):1985–1989.
39. Gern L and Rais O. Efficient transmission of Borrelia burgdorferi between cofeeding Ixodesricinus ticks (Acari: Ixodidae)[J]. Journal of Medical Entomology.1996,33(1):189–192.
40. Guerrero FD,Nene VM,George JE, et al.Sequencing a new target genome: the Boophilus microplus(Acari: Ixodidae) genome project[J]. J Med Entomol.2006,43(1):9–16.
41. Hahn P. The development of carnitine synthesis from c-butyrobetaine in the rat. Life Sci.1981,28:1057-1060.
42. Hajdusek O, Sojka D, Kopacek P, et al. Knockdown of proteins involved in iron metabolism limitstick reproduction and development[J]. Proc Natl Acad Sci U S A,2009,106(4):1033-1038.
43. Hannier S, Liversidge J, Sternberg JM, et al. Characterization of the B-cell inhibitory protein factorin Ixodes ricinus tick saliva: a potential role in enhanced Borrelia burgdorferi transmission[J].Immunology,2004,113:401–408.
44. Hill CA and Wikel SK. The Ixodes scapularis genome project: an opportunity for advancing tickresearch[J]. Trends Parasitol.2005,21(4):151–153.
45. Hoa NT,Keene KM,Olson KE, et al. Characterization of RNA interference in an Anophelesgambiae cell line[J]. Insect Biochem Mol Biol.2003,33(9):949–957.
46. Hoppel CL and Davis AT. Inter-tissue relationships in the synthesis and distribution of carnitine.Biochem Soc Trans,1986,14:673–674.
47. Humair, P.-F., Rais, O. and Gern, L.. Transmission of Borrelia afzelii from Apodemus mice andClethrionomys voles to Ixodes ricinus ticks: differential transmission pattern and overwinteringmaintenance[J]. Parasitology,1999,118:33–42.
48. Ilona A, Barash, Mathew L, et al; Muscle LIM protein plays both structural and functional roles inskeletal muscle[J]. Am J Physiol Cell Physiol,2005,289:1312-1320.
49. Jones LD, Davies CR, Steele GM,et al. A novel mode of arbovirus transmission involving anonviraemic host[J]. Science,1987,237:775–777.
50. Jones LD, Hodgson E and Nuttall PA. Enhancement of virus transmission by tick salivary glands[J].Journal of General Virology,1989,70:1895–1898.
51. Julie LK and Mary CB, The LIM domain:from the cytoskeleton to the nucleus[J].Molecular cellbiology,2004,5:921-931.
52. Karim S, Ramakrishnan VG, Tucker JS,et al.Amblyomma americanum salivary glands:double-stranded RNA-mediated gene silencing of synaptobrevin homologue and inhibition ofPGE2stimulated protein secretion[J]. Insect Biochem Mol Biol,2004,34:407–413.
53. Karim S, Ramakrishnan VG, Tucker JS, et al. Amblyomma americanum salivary gland homolog ofnSec1is essential for saliva protein secretion[J]. Biochem Biophys Res Commun,2004,324(4):1256–1263.
54. Kyckova K and Kopecky J. Effect of tick saliva on mechanisms of innate immune response againstBorrelia afzelii[J]. Journal of Medical Entomology,2006,43:1208–1214.
55. Karim S, Miller NJ, Valenzuela J, et al. RNAi-mediated gene silencing to assess the role ofsynaptobrevin and cystatin in tick blood feeding[J]. Biochem Biophys Res Commun,2005,334(4):1336–1342.
56. Kocan KM, Roman RM and de la Fuente. Transovarial silencing of the subolesin gene inthree-host ixodid tick species after injection of replete females with subolesin dsRNA. ParasitolRes,2007,100(6):1411–1415.
57. Labuda M, Austyn JM, Zuffova EK, et al. Importance of localized skin infection in tick-borneencephalitis virus transmission[J]. Virology,1996,219:357–366.
58. Labuda M, Nuttall PA, Kozuch O, et al. Nonviraemic transmission of tick-borne encephalitis virus:a mechanism for arbovirus survival in nature[J]. Experientia,1993,49:802–805.
59. Labuda M, Trimnell AR, Lickova M, et al. An antivector vaccine protects against a lethalvector-borne pathogen[J]. PLoS Pathogens,2006,2:0251–0259.
60. Lahjouji K, Mitchell GA and Qureshi IA. Carnitine transport by organic cation transporters andsystemic carnitine deficiency[J]. Mol Genet Metab,2001,73:287–297.
61. Lawrie CH, Sim RB and Nuttall PA. Investigation of the mechanisms of anti-complement activityin Ixodes ricinus ticks[J]. Molecular Immunology,2005,42:31–38.
62. Lindblad B and Lindstedt G. The mechanism of a-ketoglutarate oxidation in coupled enzymaticoxygenations[J]. Am Chem Soc,1969,91:4604-4606.
63. Lipardi C, Wei Q and Paterson BM. RNAi as random degradative PCR: siRNA primersconvertmRNAinto dsRNAs that are degraded to generate new siRNAs[J]. Cell,2001,107(3):297–307.
64. Louis HA, Pino JD, Schmeichel KL, et al. Comparison of three members of the cysteine-richprotein family reveals functional conservation and divergent patterns of gene expression[J]. J BiolChem,1997,272:27484–27491.
65. Machackova M, Obornik M and Kopecky. Effect of salivary gland extract from Ixodes ricinus tickson the proliferation of Borrelia burgdorferi sensu stricto in vivo[J]. Folia Parasitologica,2006,53:153–158.
66. Mansfield KL, Johnson N, Phipps LP, et al. Tick-borne encephalitis virus-a review of an emergingzoonosis [J]. Journal of General Virology,2009,90(8):1781–1794.
67. Ma Y,Creanga A,Lum L,et al. Prevalence of off-target effects in Drosophila RNA interferencescreens[J]. Nature,2006,443:359–363.
68. McQuiston JH, Childs JE, Chamberland ME, et al. Transmission of tick-borne agents of disease byblood transfusion: a review of known and potential risks in the United States [J]. Transfusion,2000,40(3):274-284.
69. Mello CC and Conte D. Revealing the world of RNA interference[J]. Nature,2004,431:338–342.
70. Menten CD, Couvreurb B,Joloisa O et al, Kinetic study of the antibody response during the bloodmeal of Ixodes ricinus:Implication on plasma cell maturation in vivo and for anti-Ixodesvaccination[J]. Vaccine,2011,29:2044–2050.
71. Miyoshi T, Tsuji N, Islam MK, et al. Gene silencing of a cubilin-related serine proteinase from thehard tick Haemaphysalis longicornis by RNA interference[J]. J Vet Med Sci,2004,66(1):1471–1473.
72. Mulenga A, Macaluso KR, Simser JA, et al. Dynamics of Rickettsia-tick interactions: identificationand characterization of differentially expressed mRNAs in uninfected and infected Dermacentorvariabilis[J]. Insect Mol Biol,2003,12(2):185–193.
73. Narasimhan S, Montgomery RR, DePonte K, et al. Disruption of Ixodes scapularis anticoagulationby using RNA interference[J]. Proc Natl Acad Sci,2004,101:1141–1146.
74. Nene V, Lee D, Kanga S, et al.Genes transcribed in the salivary glands of female Rhipicephalusappendiculatus ticks infected with Theileria parva[J]. Insect Biochem Mol Biol,2004,34(10):1117–1128.
75. Nijhof AM, Taoufik A, de la Fuente, et al. Gene silencing of the tick protective antigens, Bm86,Bm91and subolesin, in the one-host tick Boophilus microplus by RNA interference[J]. IntParasitol,2007,37(6):653–662.
76. Nuttall PA and Labuda M. Saliva-assisted transmission of tick-borne pathogens. In Ticks: Biology,Disease and Contro,2008, l, pp.205–219. Cambridge University Press, Cambridge, UK.
77. Nuttall PA. Displaced tick-parasite interactions at the host interface[J]. Parasitology,1998,116:65–72.
78. Ostendorff HP, Peirano RI, Peters MA, et al. Ubiquitination dependent cofactor exchange on LIMhomeodomain transcription factors[J]. Nature,2002,416(6876):99103.
79. Pal U, Li X, Wang T, et al. TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi[J].Cell,2004,119(4):457–468.
80. Parola P and Raoult D. Tick-borne bacterial diseases emerging in Europe[J]. Clin Microbiol Infect,2001,7:80–83.
81. Paul HS, Sekas G and Adibi SA. Carnitine biosynthesis in hepatic peroxisomes Demonstration ofc-butyrobetaine hydroxylase activity [J]. Eur J Biochem,1992,203:599-605
82. Pechova J, Stepanova G, Kova, et al. Tick salivary gland extract-activated transmission of Borreliaafzelii spirochaetes[J]. Parasitologica,2002,49:153–159.
83. Pedra JH, Narasimhan S, Deponte K, et al. Disruption of the salivary protein14in Ixodesscapularis nymphs and impact on pathogen acquisition[J]. Am J Trop Med Hyg,2006,75(4):677–682.
84. Ramamoorthi N, Narasimhan S, Pal U, et al. The Lyme disease agent exploits a tick protein toinfect the mammalian host[J]. Nature,2005,436:573–577.
85. Randolph SE. Tick-borne disease systems emerge from the shadows: the beauty lies in moleculardetail, the message in epidemiology[J]. Parasitology,2009,136:1403–1413.
86. Randolph SE, Gern L and Nuttall PA. Co-feeding ticks: epidemiological significance for tick-bornepathogen transmission[J]. Parasitology Today,1996,12:472–479.
87. Rebouche CJ and Seim H. Carnitine metabolism and its regulation in microorgan-isms andmammals[J]. Annu Rev Nutr,1998,18:39–61.
88. Ribeiro JM, Alarcon C, Francishetti F, et al. An annotated catalog of salivary gland transcripts fromIxodes scapularis ticks[J]. Insect Biochemistry and Molecular Biology,2006,36:111–129.
89. Riding GA, Jarmey J, McKenna RV, et al. A protective ''concealed'' antigen from Boophilusmicroplus. Purification, localization and possible function[J]. J Immunol,1994,153:5158-66.
90. Robert K, Bernhard K, Ralf W K, et al. Structure of Cysteine-and Glycine-rich Protein CRP2[J]. JBiol Chem,1998,273(36):23233–23240.
91. Roignant JY, Carre C, Mugat B, et al. Absence of transitive and systemic pathways allowscell-specific and isoform-specific RNAi in Drosophila[J]. RNA,2003,9:299–308.
92. Sadler I, Crawford AW, Michelsen JW, et al. Zyxin and cCRP: two interactive LIM domainproteins associated with the cytoskeleton[J]. J Cell Biol,1992,119(6):15731587.
93. Soares CA, Lima CM, Dolan MC, et al. Capillary feeding of specific dsRNA induces silencing ofthe isac gene in nymphal Ixodes scapularis ticks[J]. Insect Mol Biol,2005,14(4):443–452.
94. Steiber A, Kerner J and Hoppel CL. Carnitine: a nutritional, biosynthetic, and functionalperspective[J]. Mol Aspects Med,2004,25:455–473.
95. Stronach B E, Siegrist S E, Beckerle M C. Two muscle-specific LIM proteins in Drosophila[J]. JCell Biol,1996,134(5):11791195.
96. Sukumaran B, Narasimhan S, Anderson JF, et al. An Ixodes scapularis protein required for survivalof Anaplasma phagocytophilum in tick salivary glands[J]. J Exp Med.2006,203(6):1507–1517.
97. Tamai I, Ohashi R, Nezu JI, et al. Molecular and functional characterization of organiccation/carnitine transporter family in mice[J]. J Biol Chem,2000,275:40064–40072.
98. Vastenhouw, N.L. et al. Gene expression: long-term gene silencing by RNAi[J]. Nature,2006,442:882.
99. Vaz FM, Wanders RJ. Carnitine biosynthesis in mammals[J]. Biochem J,2002,361:417–429.
100. Wang G and Nuttall PA. Immunoglobulin-G binding proteins in the ixodid ticks, Rhipicephalusappendiculatus, Amblyomma variegatum and Ixodes hexagonus[J]. Parasitology,1995,111:161–165.
101. Wang H, Paesen GC and Nuttall PA. Male ticks help their mates to feed[J]. Nature,1998,391:753–754.
102. Wang X, Lee G, Liebhaber S A, et al. Human cys-teine-rich protein. A member of theLIM/double-finger family displaying coordinate serum induction with c-myc[J]. J Biol Chem,1992,267(13):91769184.
103. Wang XH, Li QL, Neeta A, et al. A role for muscle LIM protein (MLP) in vascular remodelin[J].Journal of Molecular and Cellular Cardiology,2006,40:503-509.
104. Wikel SK, Allen JR. Acquired resistance to ticks Cobra venom factor and the resistance response[J].Immunology,1977,32:457–465.
105. Wikel SK. Host immunity to ticks[J]. Annual Review of Entomology,1996,41:1–22.
106. Wikel SK, Ramachandra RN and Bergman DK. Immunological strategies for suppression of vectorarthropods: novel approaches in vector control[J]. Bull SOC Vector Ecol,1992,17:l0-19.
107. Wikswo ME, Hu R, Metzger ME, et al. Detection of Rickettsia rickettsii and Bartonella henselaein Rhipicephalus sanguineus ticks from California[J]. Journal of Medical Entomology,2007,44(1):158-162.
108. Willadsen P and McKenna RV. Vaccination with ‘concealed’ antigens: myth or reality?[J].ParasiteImmunol,1991,13:605-16.
109. Willadsen P. Tick control: thoughts on a research agenda[J]. Vet Parasitol,2006,138(3):161–168.
110. Winston WM, Molodowitch C and Hunter CP.Systemic RNAi in C. elegans requires the putativetransmembrane protein SID-1[J]. Science,2002,295(5564):2456–2459.
111. You M, Xuan X, Tsuji N, et al. Molecular characterization of a troponin I-like protein from thehard tick Haemaphysalis longicornis[J]. Insect Biochem Mol Biol,2001,32(1):67-73.
112. Zeider NS, Schneider BS, Nuncio MS, et al. Coinoculation of Borrelia spp. with tick salivary glandlysate enhances spirochete load in mice and is tick species-specific[J]. Journal of Parasitology,2002,88:1276–1278.
113. Zhang P, Tian Z, Liu G, et al.Characterization of acid phosphatase from the tick Haemaphysalislongicornis. Vet Parasitol,2011,182(2-4):287-96.
114. Zhou D, He Q, Wang C, et al.RNA interference and potential applications[J].2006, Curr Top Med.Chem.6:901–911.
115. Zhou J, Liao M, Hatta T, et al. Identification of a follistatin-related protein from the tickHaemaphysalis longicornis and its effect on tick oviposition[J]. Gene,2006,372:191–198.
2.柴慧萍，刘光远，张林，等.长角血蜱饥饿雌蜱cDNA表达文库的构建及免疫学筛选[J].中国兽医寄生虫学与寄生虫病杂志，2009，27(1)：2-10.
3.邓国藩，姜在阶.中国经济昆虫志，第39册，蜱螨亚纲硬蜱科.北京：科学出版社，1991：34-43.
4.汪明.兽医寄生虫学[M].北京：中国农业出版社，2003，75-78.
5. Alekseev AN, Chunikhin SP, Rukhkyan MY, et al. Possible role of Ixodidae salivary glandsubstrate s an adjuvant enhancing arbovirus transmission[J]. Meditsinskaya ParazitologiyaiParazitarnye Bolezni,1991,1(5):28–31.
6. Aljamali MN, Sauer JR and Essenberg RCl. RNA interference: applicability in tick research[J].Expppl Acarol,2002,28(1-4):89–96.
7. Aljamali MN, Bior AD,Sauer JR et al. RNA interference in ticks: a study using histamine bindingprotein dsRNA in the female tick Amblyomma americanum[J]. Insect Mol Biol,2003,12(3):299–305
8. Almazan C, Kocan KM, Blouin EF et al. Vaccination with recombinant tick antigens for thecontrol of Ixodes scapularis adult infestations[J]. Vaccine,23(46-47):5294–5298.
9. Almazan C, Machado UB, Kocan KM et al. Characterization of three Ixodes scapularis cDNAsprotective against tick infestations[J]. Vaccine,2005,23(35):4403–4416.
10. Anderson JF and Valenzuela JG. Tick saliva: from pharmacology and biochemistry totranscriptome analysis and functional genomes. In Ticks: Biology, Disease and Control,2008, pp.92–107.
11. Arber S, Halder G, Caroni P. Muscle LIM protein, a novel essential regulator of myogenesis,promotes myogenic differentiation[J]. Cell,1994,79(2):221231.
12. Bach I. The LIM domain: regulation by association[J]. Mech Dev,2000,91(1-2):517.
13. Bieber LL and Monroe RE.The relation of carnitine to the formation of phosphatidyl-beta-ethylcholine by Tenebrio molitor L. larvae[J]. Lipids,1969,4(4):293-8.
14. Boldbaatar D,Sikasunge CS,Battsetseg B et al. Molecular cloning and functional characterizationof an aspartic protease from the hard tick Haemaphysalis longicornis[J]. Insect Biochem MolBiol,2006,36(1):25–36
15. Bowman AS, Dillwith JW, Sauer JR. Tick Salivary prostaglandins: presence, origin andsignificance[J]. Parasitol Today,1996,12(10):388-396.
16. Bremer J. Trends in Biochem[J].Science,1997,(2):207
17. Burd CG and Dreyfuss G. Conserved structures and diversity of functions of RNA-bindingproteins[J]. Science,1994,265(5172):615621.
18. Campos E, Facanha AR, Coata EP, et al. A Mitochondrial Membrane Exopolyphosphatase IsModulated by and Plays a Role in the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus)microplus Embryos[J]. Int J Mol Sci.2011,12(6):3525-3535.
19. Cerutti, H. RNA interference: traveling in the cell and gaining functions?[J]. Trends Genet,2003,19(1):39–46.
20. Clark KA, Bland JM, Beckerle MC. The Drosophila muscle LIM protein, Mlp84B,cooperates withD-titin to maintain muscle structural integrity[J]. Journal of Cell Science,2007,120(1):2066-2077.
21. Crawford A W, Pino JD, Beckerle MC. Biochemical and molecular characterization of the chickencysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with theactin cytoskeleton[J]. J Cell Biol,1994,124(1-2):117127.
22. de la Fuente, Almazan C,Blouin EF et al. RNA interference screening in ticks for identification ofprotective antigens[J]. Parasitol Res,2005,96(3):137–141.
23. de la Fuente, Almazan C, Blouin EF et al. Reduction of tick infections with Anaplasma marginaleand A. phagocytophilum by targeting the tick protective antigen subolesin[J]. Parasitol Res,2006,100(1):85–91.
24. de la Fuente, Almazan C, Machado UB et al.The tick protective antigen,4D8, is a conservedprotein involved in modulation of tick blood ingestion and reproduction[J]. Vaccine,2006,24(19):4082–4095.
25. de la Fuente, Almazan C, Naranjo V et al. Synergistic effect of silencing the expression of tickprotective antigens4D8and Rs86in Rhipicephalus sanguineus by RNA interference[J]. ParasitolRes,2006,99(2):108–113.
26. de la Fuente, Almazan C, Naranjo V et al. Autocidal control of ticks by silencing of a single geneby RNA interference[J]. Biochem Biophys Res Commun,2006,344(1):332–338.
27. de la Fuente and Kocan KM. Strategies for development of vaccines for control of Ixodid tickspecies[J]. Parasite Immunol,2006,28(7):275–283.
28. Demma LJ, Traeger MS, Nicholson WL, et al. Rocky mountains spotted fever from an unexpectedtick vector in Arizona [J]. N Engl J Med,2005,353(6):587-594.
29. Dong Y and Friedrich M. Nymphal RNAi: systemic RNAimediated gene knockdown in juvenilegrasshopper[J]. BMC Biotechnol,2005,1472:5-25
30. Editorial. MLP: A stress sensor goes nuclear[J].Journal of Molecular and Cellular Cardiology,2009,47:423–425.
31. Faisal A and Bughdad I.Ultrastructural study of muscles fibers in Tick Hyalomma (Hyalomma)anatolicum anatolicm(Ixodoidea:Ixodidae)[J]. Pakistan Journal of Biological Sciences,2010,13(17):828-834.
32. Feinberg EH and Hunter CP. Transport of dsRNA into cells by the transmembrane protein SID-1[J].Science,2003,301(5639):1545–1547.
33. Fereira C A, Barbosa M C, Silveira T C, et al. cDNA cloning, expression and characterization ofBoophilus microplus paramyosin[J]. Parasitology,2002,125:265–274.
34. Frederic MV and Ronald JA.Carnitine biosynthesis in mammals Biochem[J],2002,361:417-429.
35. Gao JL, Luo JX, Fan R, et al. Molecular characterization of amyosin alkali light chain-like protein,a “concealed” antigen from the hard tick Haemaphysalis qinghaiensis[J].Vet Parasitol,2007,147(1-2):140–149.
36. Gao JL, Luo JX, Fan R, et al. Cloning and characterization of a cDNA clone encoding troponin Tfrom tick Haemaphysalis qinghaiensis (Acari: Ixodidae)[J]. Comp Biochem Physiol B BiochemMol Bio.2008, l151(3):323-329.
37. Garcia S,Billecocq,Crance JM, et al. Nairovirus RNA sequences expressed by a Semliki Forestvirus replicon induce RNA interference in tick cells[J]. J Virol.2005,79(14):8942–8947.
38. Garcia S, Billecocq,Crance JM, et al.Viral suppressors of RNA interference impair RNA silencinginduced by a Semliki Forest virus replicon in tick cells[J]. J Gen Virol.2006,87(7):1985–1989.
39. Gern L and Rais O. Efficient transmission of Borrelia burgdorferi between cofeeding Ixodesricinus ticks (Acari: Ixodidae)[J]. Journal of Medical Entomology.1996,33(1):189–192.
40. Guerrero FD,Nene VM,George JE, et al.Sequencing a new target genome: the Boophilus microplus(Acari: Ixodidae) genome project[J]. J Med Entomol.2006,43(1):9–16.
41. Hahn P. The development of carnitine synthesis from c-butyrobetaine in the rat. Life Sci.1981,28:1057-1060.
42. Hajdusek O, Sojka D, Kopacek P, et al. Knockdown of proteins involved in iron metabolism limitstick reproduction and development[J]. Proc Natl Acad Sci U S A,2009,106(4):1033-1038.
43. Hannier S, Liversidge J, Sternberg JM, et al. Characterization of the B-cell inhibitory protein factorin Ixodes ricinus tick saliva: a potential role in enhanced Borrelia burgdorferi transmission[J].Immunology,2004,113:401–408.
44. Hill CA and Wikel SK. The Ixodes scapularis genome project: an opportunity for advancing tickresearch[J]. Trends Parasitol.2005,21(4):151–153.
45. Hoa NT,Keene KM,Olson KE, et al. Characterization of RNA interference in an Anophelesgambiae cell line[J]. Insect Biochem Mol Biol.2003,33(9):949–957.
46. Hoppel CL and Davis AT. Inter-tissue relationships in the synthesis and distribution of carnitine.Biochem Soc Trans,1986,14:673–674.
47. Humair, P.-F., Rais, O. and Gern, L.. Transmission of Borrelia afzelii from Apodemus mice andClethrionomys voles to Ixodes ricinus ticks: differential transmission pattern and overwinteringmaintenance[J]. Parasitology,1999,118:33–42.
48. Ilona A, Barash, Mathew L, et al; Muscle LIM protein plays both structural and functional roles inskeletal muscle[J]. Am J Physiol Cell Physiol,2005,289:1312-1320.
49. Jones LD, Davies CR, Steele GM,et al. A novel mode of arbovirus transmission involving anonviraemic host[J]. Science,1987,237:775–777.
50. Jones LD, Hodgson E and Nuttall PA. Enhancement of virus transmission by tick salivary glands[J].Journal of General Virology,1989,70:1895–1898.
51. Julie LK and Mary CB, The LIM domain:from the cytoskeleton to the nucleus[J].Molecular cellbiology,2004,5:921-931.
52. Karim S, Ramakrishnan VG, Tucker JS,et al.Amblyomma americanum salivary glands:double-stranded RNA-mediated gene silencing of synaptobrevin homologue and inhibition ofPGE2stimulated protein secretion[J]. Insect Biochem Mol Biol,2004,34:407–413.
53. Karim S, Ramakrishnan VG, Tucker JS, et al. Amblyomma americanum salivary gland homolog ofnSec1is essential for saliva protein secretion[J]. Biochem Biophys Res Commun,2004,324(4):1256–1263.
54. Kyckova K and Kopecky J. Effect of tick saliva on mechanisms of innate immune response againstBorrelia afzelii[J]. Journal of Medical Entomology,2006,43:1208–1214.
55. Karim S, Miller NJ, Valenzuela J, et al. RNAi-mediated gene silencing to assess the role ofsynaptobrevin and cystatin in tick blood feeding[J]. Biochem Biophys Res Commun,2005,334(4):1336–1342.
56. Kocan KM, Roman RM and de la Fuente. Transovarial silencing of the subolesin gene inthree-host ixodid tick species after injection of replete females with subolesin dsRNA. ParasitolRes,2007,100(6):1411–1415.
57. Labuda M, Austyn JM, Zuffova EK, et al. Importance of localized skin infection in tick-borneencephalitis virus transmission[J]. Virology,1996,219:357–366.
58. Labuda M, Nuttall PA, Kozuch O, et al. Nonviraemic transmission of tick-borne encephalitis virus:a mechanism for arbovirus survival in nature[J]. Experientia,1993,49:802–805.
59. Labuda M, Trimnell AR, Lickova M, et al. An antivector vaccine protects against a lethalvector-borne pathogen[J]. PLoS Pathogens,2006,2:0251–0259.
60. Lahjouji K, Mitchell GA and Qureshi IA. Carnitine transport by organic cation transporters andsystemic carnitine deficiency[J]. Mol Genet Metab,2001,73:287–297.
61. Lawrie CH, Sim RB and Nuttall PA. Investigation of the mechanisms of anti-complement activityin Ixodes ricinus ticks[J]. Molecular Immunology,2005,42:31–38.
62. Lindblad B and Lindstedt G. The mechanism of a-ketoglutarate oxidation in coupled enzymaticoxygenations[J]. Am Chem Soc,1969,91:4604-4606.
63. Lipardi C, Wei Q and Paterson BM. RNAi as random degradative PCR: siRNA primersconvertmRNAinto dsRNAs that are degraded to generate new siRNAs[J]. Cell,2001,107(3):297–307.
64. Louis HA, Pino JD, Schmeichel KL, et al. Comparison of three members of the cysteine-richprotein family reveals functional conservation and divergent patterns of gene expression[J]. J BiolChem,1997,272:27484–27491.
65. Machackova M, Obornik M and Kopecky. Effect of salivary gland extract from Ixodes ricinus tickson the proliferation of Borrelia burgdorferi sensu stricto in vivo[J]. Folia Parasitologica,2006,53:153–158.
66. Mansfield KL, Johnson N, Phipps LP, et al. Tick-borne encephalitis virus-a review of an emergingzoonosis [J]. Journal of General Virology,2009,90(8):1781–1794.
67. Ma Y,Creanga A,Lum L,et al. Prevalence of off-target effects in Drosophila RNA interferencescreens[J]. Nature,2006,443:359–363.
68. McQuiston JH, Childs JE, Chamberland ME, et al. Transmission of tick-borne agents of disease byblood transfusion: a review of known and potential risks in the United States [J]. Transfusion,2000,40(3):274-284.
69. Mello CC and Conte D. Revealing the world of RNA interference[J]. Nature,2004,431:338–342.
70. Menten CD, Couvreurb B,Joloisa O et al, Kinetic study of the antibody response during the bloodmeal of Ixodes ricinus:Implication on plasma cell maturation in vivo and for anti-Ixodesvaccination[J]. Vaccine,2011,29:2044–2050.
71. Miyoshi T, Tsuji N, Islam MK, et al. Gene silencing of a cubilin-related serine proteinase from thehard tick Haemaphysalis longicornis by RNA interference[J]. J Vet Med Sci,2004,66(1):1471–1473.
72. Mulenga A, Macaluso KR, Simser JA, et al. Dynamics of Rickettsia-tick interactions: identificationand characterization of differentially expressed mRNAs in uninfected and infected Dermacentorvariabilis[J]. Insect Mol Biol,2003,12(2):185–193.
73. Narasimhan S, Montgomery RR, DePonte K, et al. Disruption of Ixodes scapularis anticoagulationby using RNA interference[J]. Proc Natl Acad Sci,2004,101:1141–1146.
74. Nene V, Lee D, Kanga S, et al.Genes transcribed in the salivary glands of female Rhipicephalusappendiculatus ticks infected with Theileria parva[J]. Insect Biochem Mol Biol,2004,34(10):1117–1128.
75. Nijhof AM, Taoufik A, de la Fuente, et al. Gene silencing of the tick protective antigens, Bm86,Bm91and subolesin, in the one-host tick Boophilus microplus by RNA interference[J]. IntParasitol,2007,37(6):653–662.
76. Nuttall PA and Labuda M. Saliva-assisted transmission of tick-borne pathogens. In Ticks: Biology,Disease and Contro,2008, l, pp.205–219. Cambridge University Press, Cambridge, UK.
77. Nuttall PA. Displaced tick-parasite interactions at the host interface[J]. Parasitology,1998,116:65–72.
78. Ostendorff HP, Peirano RI, Peters MA, et al. Ubiquitination dependent cofactor exchange on LIMhomeodomain transcription factors[J]. Nature,2002,416(6876):99103.
79. Pal U, Li X, Wang T, et al. TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi[J].Cell,2004,119(4):457–468.
80. Parola P and Raoult D. Tick-borne bacterial diseases emerging in Europe[J]. Clin Microbiol Infect,2001,7:80–83.
81. Paul HS, Sekas G and Adibi SA. Carnitine biosynthesis in hepatic peroxisomes Demonstration ofc-butyrobetaine hydroxylase activity [J]. Eur J Biochem,1992,203:599-605
82. Pechova J, Stepanova G, Kova, et al. Tick salivary gland extract-activated transmission of Borreliaafzelii spirochaetes[J]. Parasitologica,2002,49:153–159.
83. Pedra JH, Narasimhan S, Deponte K, et al. Disruption of the salivary protein14in Ixodesscapularis nymphs and impact on pathogen acquisition[J]. Am J Trop Med Hyg,2006,75(4):677–682.
84. Ramamoorthi N, Narasimhan S, Pal U, et al. The Lyme disease agent exploits a tick protein toinfect the mammalian host[J]. Nature,2005,436:573–577.
85. Randolph SE. Tick-borne disease systems emerge from the shadows: the beauty lies in moleculardetail, the message in epidemiology[J]. Parasitology,2009,136:1403–1413.
86. Randolph SE, Gern L and Nuttall PA. Co-feeding ticks: epidemiological significance for tick-bornepathogen transmission[J]. Parasitology Today,1996,12:472–479.
87. Rebouche CJ and Seim H. Carnitine metabolism and its regulation in microorgan-isms andmammals[J]. Annu Rev Nutr,1998,18:39–61.
88. Ribeiro JM, Alarcon C, Francishetti F, et al. An annotated catalog of salivary gland transcripts fromIxodes scapularis ticks[J]. Insect Biochemistry and Molecular Biology,2006,36:111–129.
89. Riding GA, Jarmey J, McKenna RV, et al. A protective ''concealed'' antigen from Boophilusmicroplus. Purification, localization and possible function[J]. J Immunol,1994,153:5158-66.
90. Robert K, Bernhard K, Ralf W K, et al. Structure of Cysteine-and Glycine-rich Protein CRP2[J]. JBiol Chem,1998,273(36):23233–23240.
91. Roignant JY, Carre C, Mugat B, et al. Absence of transitive and systemic pathways allowscell-specific and isoform-specific RNAi in Drosophila[J]. RNA,2003,9:299–308.
92. Sadler I, Crawford AW, Michelsen JW, et al. Zyxin and cCRP: two interactive LIM domainproteins associated with the cytoskeleton[J]. J Cell Biol,1992,119(6):15731587.
93. Soares CA, Lima CM, Dolan MC, et al. Capillary feeding of specific dsRNA induces silencing ofthe isac gene in nymphal Ixodes scapularis ticks[J]. Insect Mol Biol,2005,14(4):443–452.
94. Steiber A, Kerner J and Hoppel CL. Carnitine: a nutritional, biosynthetic, and functionalperspective[J]. Mol Aspects Med,2004,25:455–473.
95. Stronach B E, Siegrist S E, Beckerle M C. Two muscle-specific LIM proteins in Drosophila[J]. JCell Biol,1996,134(5):11791195.
96. Sukumaran B, Narasimhan S, Anderson JF, et al. An Ixodes scapularis protein required for survivalof Anaplasma phagocytophilum in tick salivary glands[J]. J Exp Med.2006,203(6):1507–1517.
97. Tamai I, Ohashi R, Nezu JI, et al. Molecular and functional characterization of organiccation/carnitine transporter family in mice[J]. J Biol Chem,2000,275:40064–40072.
98. Vastenhouw, N.L. et al. Gene expression: long-term gene silencing by RNAi[J]. Nature,2006,442:882.
99. Vaz FM, Wanders RJ. Carnitine biosynthesis in mammals[J]. Biochem J,2002,361:417–429.
100. Wang G and Nuttall PA. Immunoglobulin-G binding proteins in the ixodid ticks, Rhipicephalusappendiculatus, Amblyomma variegatum and Ixodes hexagonus[J]. Parasitology,1995,111:161–165.
101. Wang H, Paesen GC and Nuttall PA. Male ticks help their mates to feed[J]. Nature,1998,391:753–754.
102. Wang X, Lee G, Liebhaber S A, et al. Human cys-teine-rich protein. A member of theLIM/double-finger family displaying coordinate serum induction with c-myc[J]. J Biol Chem,1992,267(13):91769184.
103. Wang XH, Li QL, Neeta A, et al. A role for muscle LIM protein (MLP) in vascular remodelin[J].Journal of Molecular and Cellular Cardiology,2006,40:503-509.
104. Wikel SK, Allen JR. Acquired resistance to ticks Cobra venom factor and the resistance response[J].Immunology,1977,32:457–465.
105. Wikel SK. Host immunity to ticks[J]. Annual Review of Entomology,1996,41:1–22.
106. Wikel SK, Ramachandra RN and Bergman DK. Immunological strategies for suppression of vectorarthropods: novel approaches in vector control[J]. Bull SOC Vector Ecol,1992,17:l0-19.
107. Wikswo ME, Hu R, Metzger ME, et al. Detection of Rickettsia rickettsii and Bartonella henselaein Rhipicephalus sanguineus ticks from California[J]. Journal of Medical Entomology,2007,44(1):158-162.
108. Willadsen P and McKenna RV. Vaccination with ‘concealed’ antigens: myth or reality?[J].ParasiteImmunol,1991,13:605-16.
109. Willadsen P. Tick control: thoughts on a research agenda[J]. Vet Parasitol,2006,138(3):161–168.
110. Winston WM, Molodowitch C and Hunter CP.Systemic RNAi in C. elegans requires the putativetransmembrane protein SID-1[J]. Science,2002,295(5564):2456–2459.
111. You M, Xuan X, Tsuji N, et al. Molecular characterization of a troponin I-like protein from thehard tick Haemaphysalis longicornis[J]. Insect Biochem Mol Biol,2001,32(1):67-73.
112. Zeider NS, Schneider BS, Nuncio MS, et al. Coinoculation of Borrelia spp. with tick salivary glandlysate enhances spirochete load in mice and is tick species-specific[J]. Journal of Parasitology,2002,88:1276–1278.
113. Zhang P, Tian Z, Liu G, et al.Characterization of acid phosphatase from the tick Haemaphysalislongicornis. Vet Parasitol,2011,182(2-4):287-96.
114. Zhou D, He Q, Wang C, et al.RNA interference and potential applications[J].2006, Curr Top Med.Chem.6:901–911.
115. Zhou J, Liao M, Hatta T, et al. Identification of a follistatin-related protein from the tickHaemaphysalis longicornis and its effect on tick oviposition[J]. Gene,2006,372:191–198.