小亚璃眼蜱唾液腺cDNA表达文库构建及4D8基因的克隆与原核表达
|
摘要
小亚璃眼蜱(Hyalomma anatolicum anatolicum)是一种重要的外寄生虫,在国内主要分布于新疆自治区的半荒漠草原地区,除可通过叮咬吸血使宿主生产性能降低外,还可作为许多疾病的传播媒介,是制约流行区畜牧业发展、危害居民身体健康的重要因素。
本研究采用分子生物学方法构建了小亚璃眼蜱成蜱半饱血雌蜱唾液腺cDNA表达文库,首先从小亚璃眼蜱成蜱半饱血雌蜱唾液腺中提取总RNA,并分离纯化mRNA,采用oligo(dT)引物合成双链cDNA,在其两端加EcoRⅠ/HindⅢ定向接头,将所产生的cDNA分子用Mini Column Fractionation凝胶过滤柱纯化后定向克隆到λSCREEN载体的EcoRⅠ和HindⅢ的双酶切位点之间,体外包装后得到小亚璃眼蜱唾液腺cDNA表达文库。经测定,所构建的表达文库的容量为4.0×105pfu/mL,扩增后的文库库容量为8.8×109pfu/mL,重组率为95%,这说明所构建小亚璃眼蜱唾液腺cDNA表达文库库容量大、重组率高。
根据GenBank已公布的边缘革蜱4D8基因序列设计引物,采用PCR技术自小亚璃眼蜱成蜱半饱血雌蜱唾液腺cDNA表达文库中,扩增获得了4D8基因;将其连入到pMD-18-T载体,构建重组克隆载体pMD-18-Hyaa4D8,测序分析表明,克隆的小亚璃眼蜱4D8基因与GenBank上公布的边缘革蜱4D8基因的核苷酸和氨基酸序列的同源性分别为89.2%和96.0%。然后对重组克隆载体pMD-18-Hyaa4D8进行双酶切,获得带有粘性末端的4D8基因片段,并将此片段定向亚克隆到原核表达载体pGEX-4T-1,成功构建重组原核表达载体pGEX-4T-1-Hyaa4D8。将其转化到BL21宿主菌中,用IPTG进行诱导表达,经SDS-PAGE检测,表达产物为分子量为45 ku的融合蛋白,其表达蛋白浓度为1.24 mg/mL,目的蛋白约占菌体总蛋白的33.0%。采用Western-blotting实验,分析表明此表达产物能被小亚璃眼蜱唾液腺免疫兔血清所识别。
Hyalomma anatolicum anatolicum is an ectoparasite which is distributed mainly in Uygur autonomous region. It can transmit many species of haemoprotozoa parasites and can cause the decrease in the productivity by biting and sucking of animal blood. It inhibits the development of animal husbandry and threatens the inhabitant health.
The cDNA expression library of salivary gland from female Hyalomma anatolicum anatolicum was constructed. In order to clone and study functional genes of Hyalomma anatolicum anatolicum. Total RNA were extracted by salivary gland of Hyalomma anatolicum anatolicum and mRNA were further purified. A library of oligo(dT)-primed cDNA with directional EcoRⅠ/ HindⅢlinkers was synthesized and purified by Mini Column Fractionation Kit, then ligated with theλscreen vector contain EcoRⅠ/ HindⅢarms. After package in vitro, the cDNA library of salivary gland of Hyalomma anatolicum anatolicum were constructed and amplified, The cDNA primary library titer and amplification library are 4.0×105 pfu/mL and 8.8×109 pfu/mL,respectively.
Specific primers were designed according to the gene 4D8 sequence of Dermacentor marginatum in GenBank. The gene 4D8 was amplified by PCR from cDNA expression library of salivary gland from female Hyalomma anatolicum anatolicum and cloned into pMD-18-T vector. Sequencing result showed that the nucleotide sequence and amino acid sequence of the cloned 4D8 gene shared 89.2% and 96.0% homology with the data published in GenBank respectively. A recombinant expression plasmid pGEX-4T-1-Hyaa4D8 was constructed by sub-cloning 4D8 fragment digested from plasmid pMD-18-Hyaa4D8 into linearized pGEX-4T-1 vector. The plasmid pGEX-4T-1-Hyaa4D8 was expressed in E. coli induced by IPTG. SDS- PAGE showed that a fusion protein with a molecular weight of 45ku was expressed. The concentration of expressed 4D8 protein was 1.24mg/mL. The gene 4D8 expressed level could reach up to 33.0% of total E. coli proteins.
The recombinant fusion protein could be detected by rabbit anti-Hyalomma anatolicum anatolicum positive serum with the Western-blotting.
本研究采用分子生物学方法构建了小亚璃眼蜱成蜱半饱血雌蜱唾液腺cDNA表达文库,首先从小亚璃眼蜱成蜱半饱血雌蜱唾液腺中提取总RNA,并分离纯化mRNA,采用oligo(dT)引物合成双链cDNA,在其两端加EcoRⅠ/HindⅢ定向接头,将所产生的cDNA分子用Mini Column Fractionation凝胶过滤柱纯化后定向克隆到λSCREEN载体的EcoRⅠ和HindⅢ的双酶切位点之间,体外包装后得到小亚璃眼蜱唾液腺cDNA表达文库。经测定,所构建的表达文库的容量为4.0×105pfu/mL,扩增后的文库库容量为8.8×109pfu/mL,重组率为95%,这说明所构建小亚璃眼蜱唾液腺cDNA表达文库库容量大、重组率高。
根据GenBank已公布的边缘革蜱4D8基因序列设计引物,采用PCR技术自小亚璃眼蜱成蜱半饱血雌蜱唾液腺cDNA表达文库中,扩增获得了4D8基因;将其连入到pMD-18-T载体,构建重组克隆载体pMD-18-Hyaa4D8,测序分析表明,克隆的小亚璃眼蜱4D8基因与GenBank上公布的边缘革蜱4D8基因的核苷酸和氨基酸序列的同源性分别为89.2%和96.0%。然后对重组克隆载体pMD-18-Hyaa4D8进行双酶切,获得带有粘性末端的4D8基因片段,并将此片段定向亚克隆到原核表达载体pGEX-4T-1,成功构建重组原核表达载体pGEX-4T-1-Hyaa4D8。将其转化到BL21宿主菌中,用IPTG进行诱导表达,经SDS-PAGE检测,表达产物为分子量为45 ku的融合蛋白,其表达蛋白浓度为1.24 mg/mL,目的蛋白约占菌体总蛋白的33.0%。采用Western-blotting实验,分析表明此表达产物能被小亚璃眼蜱唾液腺免疫兔血清所识别。
Hyalomma anatolicum anatolicum is an ectoparasite which is distributed mainly in Uygur autonomous region. It can transmit many species of haemoprotozoa parasites and can cause the decrease in the productivity by biting and sucking of animal blood. It inhibits the development of animal husbandry and threatens the inhabitant health.
The cDNA expression library of salivary gland from female Hyalomma anatolicum anatolicum was constructed. In order to clone and study functional genes of Hyalomma anatolicum anatolicum. Total RNA were extracted by salivary gland of Hyalomma anatolicum anatolicum and mRNA were further purified. A library of oligo(dT)-primed cDNA with directional EcoRⅠ/ HindⅢlinkers was synthesized and purified by Mini Column Fractionation Kit, then ligated with theλscreen vector contain EcoRⅠ/ HindⅢarms. After package in vitro, the cDNA library of salivary gland of Hyalomma anatolicum anatolicum were constructed and amplified, The cDNA primary library titer and amplification library are 4.0×105 pfu/mL and 8.8×109 pfu/mL,respectively.
Specific primers were designed according to the gene 4D8 sequence of Dermacentor marginatum in GenBank. The gene 4D8 was amplified by PCR from cDNA expression library of salivary gland from female Hyalomma anatolicum anatolicum and cloned into pMD-18-T vector. Sequencing result showed that the nucleotide sequence and amino acid sequence of the cloned 4D8 gene shared 89.2% and 96.0% homology with the data published in GenBank respectively. A recombinant expression plasmid pGEX-4T-1-Hyaa4D8 was constructed by sub-cloning 4D8 fragment digested from plasmid pMD-18-Hyaa4D8 into linearized pGEX-4T-1 vector. The plasmid pGEX-4T-1-Hyaa4D8 was expressed in E. coli induced by IPTG. SDS- PAGE showed that a fusion protein with a molecular weight of 45ku was expressed. The concentration of expressed 4D8 protein was 1.24mg/mL. The gene 4D8 expressed level could reach up to 33.0% of total E. coli proteins.
The recombinant fusion protein could be detected by rabbit anti-Hyalomma anatolicum anatolicum positive serum with the Western-blotting.
引文
[1].汪明主编,兽医寄生虫学[M].中国农业出版社,2003,第三版,北京,75-88.
[2].孔繁瑶主编.家畜寄生虫学[M].北京:农业出版社,1981,293-296.
[3].邓国藩编著.中国经济昆虫志[M].北京:第十五册,蜱螨目蜱总科,科学出版社,1978,2.
[4].邓国藩,姜在阶主编.中国经济昆虫志[M].北京:第三十九册,蜱螨亚纲硬蜱科.科学出版社,1991,2.
[5]. Balashov,Y.S. Bloodsucking tick(Ixodoidea)-vector of disease of man and animals (English translation)[J].Misc.Publ.Entomol. Soc. Amer.1972,8:163-376.
[6].于心,叶瑞玉,龚正达.新疆蜱类志[M].乌鲁木齐:新疆科学技术卫生出版社.1997,79.
[7].阿布力克木·阿不都热西提,李冰,蒋卫等.吐鲁番地区首次发现小亚璃眼蜱的报告[J].地方病通报,2006,6 (21) : 10-11.
[8].罗建勋,殷宏,关贵全,等.小亚璃眼蜱对牛巴贝斯虫未定种和环形泰勒虫传播的试验研究[J].畜牧兽医学报,2005,36 (11):1197-1201.
[9]. Luo J X , Yin H , Guan G Q , et al. Description of new B abesia sp. infective for cattle in China [J]. Parasitology Research,2002,88 : S13–S15.
[10].罗建勋,殷宏,刘光远,关贵全,等.牛的巴贝斯虫18S rRNA基因序列比较研究[J].畜牧兽医学报,2005,36 (9):906-911.
[11].薄新文,汪明.宿主对蜱免疫的研究进展[J].中国兽医杂志,1998,24(10):45-47.
[12].袁改玲.青海血蜱生活史及不同发育阶段蛋白分析[D].呼和浩特,内蒙古农业大学,2002.
[13]. Wikel SK., Alarcon-Chaidez F.J. Progress toward molecular caracterization of ectoparasite moduoation of host immunity[J].Veterinary Parasitolog,2001,101: 275-287.
[14]. Benjamin L.HARI. Role of crooming in biological control of ticks[J]. Annals of the new academy of sciences, 2000,12:565-569.
[15].赵忠芳,孔繁瑶等.兔非特异性免疫力抗长角血蜱幼虫感染能力的研究[J].中国农业大学学报, 1998,(增刊):79-81.
[16].童竞亚主编.医学免疫学与微生物学[M].北京:人民卫生出版社,1993.
[17]. Johnston LAY, Kemp DH. Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks. Effects of induced immunity on tick populations [J]. International Journal for Parasitology,1986, 16: 27-34.
[18]. Kemp DH,Agbede RIS,Johnston LAY.Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: feeding and survival of the parasite on vaccinated cattle [J]. International Journal for Parasitology, 1986, 16: 115.
[19]. Opdebeeck J.P., Wong J.Y.M., Jackson L.A. Vaccines to protect Hereford cattle againstthe cattle tick, Boophilus microplus [J]. Immunology, 988, 63: 363.
[20]. Willadsen P, Riding GA, Mckenna RV. Immunologic Control of A Parasitic arthropod: Identification of a Protective Antigen from Boophilus microplus [J]. The Journal of Immunology, 1989, 143(4): 1346-1351.
[21]. Willadsen P, Eisemann CH, Tellam RL.“Concealed”antigens: expanding the range of immunological targets [J]. Parasitol Today, 1993, 9: 132.
[22]. Tsuda A, Mulenga A, Sugimoto C. cDNA cloning, characterization and vaccine effect analysis of Haemaphysalis longicornis tick saliva protein [J]. Vaccine, 2001, 19: 4287-4296.
[23]. Tsuji N, Kamio T, Isobe T. Molecular characterization of a peroxiredoxin from the hard tick Haemaphysalis longicornis[J]. Insect Molecular Biology, 2001, 10(2): 121-129.
[24]. Willadsen P., Riding G.A., Mckenna R.V., et al. Immunologic Control of A Parasitic Arthropod: Identification of a Protective Antigen from Boophilus microplus [J].The Journal of Immunology, 1989,143(4): 1346-1351.
[25]. Penichet M., Rodriguez M., Castellano O., et al. Detection of Bm86 antigen in different strains of Boophilus microplus and effectiveness of immunization with recombinant Bm86 [J]. Parasite Immunology, 1994, 16: 493-500.
[26]. Rand K.N., Moore T., Sriskantha A., et al. Cloning and expression of a protective antigen from the cattle tick Boophilus microplus [J]. Proc. Natl. Acad. Sci. USA. 1989, 86: 9657-9661.
[27]. Turnbull I.F., Smith D.R.J., Sharp P.J., et al. Expression and Secretion in Aspergillus nidulans and Aspergillus niger of a Cell Surface Glycoprotein from the Cattle Tick, Boophilus microplus, by Using the Fungal amdS Promoter System[J]. Applied and Environmental Microbiology, 1990, 56(9): 2847-2852.
[28]. Rodríguez M., Rubiera R., Penichet M., et al. High level expression of the B.micorplus Bm86 antigen in the yeast P.pastoris forming highly immunogenic particles for cattle [J]. Biotechnol, 1994, 33: 135-146.
[29]. Montesino R., Cremata J., Rodríguez M., et al. Biochemical characterization of the recombinant Boophilus microplus Bm86 antigen expressed by transformed[J]. Pichia pastoris cells. Biotechnol. Appl. Biochem, 1996, 23: 23-28.
[30]. García-García J.C., Montero C., Rodríguez M., et al. Effect of particulation on the immunogenic and protective properties of the recombinant Bm86 antigen expressed in Pichia pastoris [J]. Vaccine,1998, 16(4): 374-381.
[31]. García-García J.C., Montero C., Redondo M., et al. Control of ticks resistant to immunization with Bm86 in cattle vaccinated with the recombinant antigen Bm95 isolated from the cattle tick, Boophilus microplus[J]. Vaccine. 2000, 18: 2275-2287.
[32]. Riding G.A., Jarmey J., McKenna R.V., et al. A Protective“concealed”Antigen fromBoophilus microplus: Purification, Localization, and Possible Function [J]. The Journal of Immunology, 1994, 153: 5158-5166.
[33]. McKenna R.V., Riding G.A., Jarmey J.M., et al. Vaccination of cattle against the Boophilus microplus using a mucin-like membrane glycoprotein[J]. Parasite Immunology, 1998, 20: 325-336.
[34]. Rose R.D., McKenna R.V., Cobon G., et al. Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep[J]. Veterinary Immunology and Immunopathology, 1999, 71: 151-163.
[35]. Lee R.P., Opdebeeck J.P. Antigens Identified by Monoclonal Antibodies in Tissue Sections of Boophilus microplus [J]. International Journal for Parasitology, 1995, 25(2): 241-248.
[36]. Lee R.P., Opdebeeck J.P. Isolation of protective antigens from the gut of Boophilus microplus using monoclonal antibodies [J]. Immunology, 1991, 72: 121-126.
[37]. Toro-Ortiz R.D., et al. Monoclonal antibodies against Boophilus microplus and their effects on tick reproductive efficiency [J]. Veterinary Parasitology,1997, 69: 297-306.
[38].樊瑞泉,罗建勋,杨孝朴,殷宏.微小牛蜱Bm86基因的克隆与原核表达[J].甘肃农业大学学报,2007,42(1):15-19.
[39].李文卉,罗建勋,殷宏,等.微小牛蜱Bm86基因的真核表达[J].中国兽医科技,2005,35(9):718-722.
[40]. Mulenga A., Sugimoto C., Sako Y., et al. Molecular Characterization of a Haemaphysalis longicornis Tick Salivary Gland-Associated 29-Kilodalton Protein and Its Effect as a Vaccine against Tick Infestation in Rabbits[J]. Infection and Immunity,1999, 67(4): 1652-1658.
[41]. Tsuda A, Mulenga A, Sugimoto C, et al. cDNA cloning, characterization and vaccine effect analysis of Haemaphysalis longicornis tick saliva protein[J]. Vaccine, 2001, 19: 4287-4296.
[42]. You M., Xuan X., Tsuji N., et al. Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis[J]. Insect Biochemistry and Molecular Biology ,2001, 32: 67-73.
[43]. Tsuda A., Mulenga A., Sugimoto C., et al. cDNA cloning, characterization and vaccine effect analysis of Haemaphysalis longicornis tick saliva protein[J]. Vaccine, 2001, 19: 4287-4296.
[44]. Neurath H. The versatility of proteolytic enzymes. J Cell Bioche,1986, 32: 35-49
[45]. Rao M.B., Tanksale A.M., et al. Molecular and biotechnology aspects of microbial proteases [J]. Microb. Mol. Biol. Rev,1998, 62: 597-635.
[46]. Mulenga M., Sugino M., Nakajima M., et al. Tick-encoded serine proteinase inhibitors (serpins): Potential target antigens for tick vaccine development [J]. J. Vet. Med. Sci, 2001a, 63: 1063-1069.
[47]. Liao M, Zhou J, Hatta T, et al. Molecular characterization of Rhipicephalus(Boophilus) microplusBm86 homologue from Haemaphysalis longicornis ticks[J]. VetParasitol. 2007, 11(2): 146-157.
[48].高金亮.青海血蜱CDNA表达文库的构建和隐藏抗原的筛选、克隆与表达[D].北京：中国农业科院研究生院，2004.
[49].赵海平.青海血蜱幼蜱CDNA表达文库的构建及免疫学筛选[D].北京：中国农业科院研究生院，2007.
[50]. S. Ghosh N. K. Singh G. Das .Assessment of duration of immunity in crossbredcattle immunized with glycoproteins isolated from Hyalomma anatolicum anatolicumand Boophilus microplus[J].Parasitol Res, 2005, 95: 319–326.
[51]. Ghosh S, Khan MH. Isolation of immunoreactive proteins from the larval extracts ofBoophilus microplus and Hyalomma anatolicum anatolicum[J].Indian J Anim Sci,2000a, 70: 14–18.
[52]. Leboulle.G,Rochez C,Louahed J, et al. Ixodes ricinus salivary gland mRNAencoding factors induced during blood feeding[J].Am J Trop Med Hvg, 2002, 6(3):225-233.
[53]. Gill H S , Boid R , Ross C A. Isolation and characterization of salivary antigens fromHyalom ma anatolicum anatolicum [J]. ParasiteImmunology, 1986, 8: 11225.
[54]. Sran H.S.,Grewal A.S.,Kondal J.K. et al. Enhanced immunity to Hyalommaanatolicum anatolicum ticks in cross-bred calves using the tick salivary glandextractantigens[J].Veterinary Immunologyand Immunopathology, 1996, 51: 333-343.
[55]. Sharma,J.K.,Ghosh, et al Immunoprotective efficacy of a purified 39kDa nymphalantigen of Hyalomma anatolicum anatolicum[J]. Vaccine, 2001, 33(2): 103-116.
[56]. Kaba SA, Musoke AJ, Schaap D, et al. Novel baculovirus derived p67 subunitvaccines efficacious against East Coast fever in cattle[J]. Vaccine, 2005,23:2791–2800.
[57]. Musoke A, Rowlands J, Nene V, et al. Subunit vaccine based on the p67 majorsurface protein of Theileria parva sporozoites reduces severity of infection derivedfrom field tick challenge [J].Vaccine , 2005, 23: 3084–3095.
[58]. S. Ghosh P. Azhahianambi Jose, de la Fuente. Control of ticks of ruminants, withspecial emphasis on livestock farming systems in India: present and future possibilitiesfor integrated control—a review [J]. Exp Appl Acarol, 2006, 40: 49–66.
[59]. SANDER DE VOS1, LAURA ZEINSTRA1, OMAR TAOUFIK1, et al. Evidence forthe utility of the Bm86 antigen from Boophilus microplus in vaccination against othertick species [J]. Experimental and Applied Acarology , 2001,25: 245–261.
[60]. Mulenga A., Tsuda A., Onuma M., et al. Four serine proteinase inhibitors (serpin) fromthe brown ear tick, Rhiphicephalus appendiculatus: cDNA cloning and preliminarycharacterization[J]. Insect Biochemistry and Molecular Biology, 2003, 33: 267-276.
[61]. Kopacek P., Zdychova J., Yoshiga T., et al. Molecular cloning, expression and isolationof ferritins from two tick species- Ornithodoros moubata and Ixodes ricinus[J]. Insect Biochem Mol Biol, 2003, 33: 103-113.
[62].周金林.蜱的功能分子研究及其应用前景.动物医学进展[J]. 2004,52(1):53-56.
[63]. David Odongo, Lucy Kamau, Robert Skilton, et al. Vaccination of cattle with TickGARD induces cross-reactive antibodies binding to conserve Boophilus decoloratus[J]. Vaccine,2007,25: 1287–1296.
[64]. Almaza′n C, Kocan KM, Bergman DK, et al. Identification of protective antigens for the control of Ixodes scapularis infestations using cDNA expression library immunization [J]. Vaccine, 2003a, 21: 1492–1501.
[65]. Almaza′n C, Kocan KM, Bergman DK, et al. Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of expressed sequence tags [J]. Gene Ther Mol Biol , 2003b, 7: 43–59.
[66]. Almaza′n C, Blas-Machado U, Kocan KM, et al. Characterization of three Ixodes scapularis cDNAs protective against tick infestations [J].Vaccine,2005a,23: 4403–4416.
[67]. Almaza′n C, Kocan KM, Blouin EF, et al. Vaccination with recombinant tick antigens for the control of Ixodes scapularis adult infestations[J].Vaccine,2005b,23: 5294–5298.
[68]. de la Fuente J, Kocan KM . Strategies for development of vaccines for control of ixodid tick species [J]. Parasite Immunol, 2006, 28: 275–286.
[69]. Nijhof AM, Taoufik A, de la Fuente J, et al. Gene silencing of the tick protective antigens,Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference [J]. Int J Parasitol, 2006.10: 1015-1017.
[70]. Kocan KM.The tick protective antigen, 4D8, is a conserved protein involved in modulation of tick blood digestion and reproduction[J].Vaccine, 2006, 24: 4082–4095.
[71]. Katherine M. Kocan. Raúl Manzano-Roman,et al.Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA[J]. Parasitol Res, 2007, 100: 1411–1415.
[72]. Aljamali MN, Sauer JR, Essenberg RC. RNA interference applicability in tick research[J]. Exp Appl Acarol, 2002, 28: 89–96.
[73]. Almazán C, Kocan KM, Bergman DK, et al. Identification of protective antigens for the control of Ixodes scapularis infestations using cDNA expression library immunization[J]. Vaccine ,2003, 21: 1492–1501.
[74]. Almazán C,Blas-Machado U,Kocan KM,et al.Characterization of three Ixodes scapularis cDNAs protective against tick infestations[J].Vaccine, 2005,23: 4403–4416.
[75]. de la Fuente J, Kocan KM. Strategies for development of vaccines for control of ixodid tick species[J]. Parasite Immunol, 2006, 28: 275–283.
[76]. de la Fuente J, Almazán C, Blouin EF, et al. RNA interference screening in ticks for identification of protective antigens[J]. Parasitol Res, 2005, 96: 137–141.
[77]. de la Fuente J, Kocan KM . Advances in the identification and characterization of protective antigens for recombinant vaccines against tick infestations[J]. Expert Rev Vaccines, 2003, 2: 583–593.
[78]. de la Fuente J, Almazán C, Naranjo V, et al. Synergistic effect of silencing the expression tick protective antigens 4D8 and Rs86 in Rhipicephalus sanguineus by RNA interference[J]. Parasitol Res, 2006b, 99: 108–113.
[79]. de la Fuente J, Almazán C, Blouin EF, et al. Reduction of tick infections with Anaplasma marginale and A. phagocytophilum by targeting the tick protective antigen subolesin[J]. Parasitol Res, 2006d, 100: 85–91.
[80]. Taoufik Amar, Nijhof Ard M, de la Fuente, et al. Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference[J]. International Journal for Parasitology, 2006, 11: 5.
[81]. Willadsen P., Bird P., Cobon G.S., et al. Commercialisation of a recombinant vaccine against Boophilus microplus[J]. Parasitology, 1995, 110: S43-S50.
[82]. Tellam R.L., Smith D., et al. Vaccination against ticks[J]. In Animal Parasite Control Utilizing Biotechnology, 1992, 12: 303-331.
[83]. Rodriguez M., Penichet M.L., Mouris A.E., et al. Control of Boophilus mircoplus population in grazing cattle vaccinated with a recombinant Bm86 antigen preparation[J]. Vet parasitol,1995a, 57: 339-349.
[84]. Canales M., Enríquez A., Ramos E., et al. Large-scale production in Pichia Pastoris of the recombinant vaccine Gavac? against cattle tick[J]. Vaccine. 1997, 15(4): 414-422
[85]. Rodríguez M., Massard C.L., Fonseca A.H.D., et al. Effect of vaccination with a recombinant Bm86 antigen preparation on natural infestations of Boophilus microhlus in grazing dairy and beef pure and cross-breed cattle in Brazil[J]. Vaccine, 1995b, 13(18): 1804-1808.
[86]. Rodríguez M., Montero C., et al. Effect of vaccination with Gavac? on the incidence of Babesia bovis infections and the reduction in the number and frequency of acaricide treatments in cattle under production conditions in Cuba. In: Recombinant Vaccines for the Control of Cattle Tick[J]. Elfos Scientiae, La Habana, Cuba, 1995c, 187-194.
[87]. Vanegas L.F., Parra S.A., et al. Commercialization of the recombinant Vaccine Gavac? against Boophilus microplus in Colombia. In: Recombinant Vaccines for the Control of Cattle Tick[J]. Elfos Scientiae, La Habana, Cuba, 1995, 196-199.
[88]. Massard C.L., Fonseca R.N., et al. Effect of Vaccination with Gavac? on the reduction in the number and frequency of acaricide treatments in cattle under production conditions in Brazil. In: Recombinant Vaccines for the Control of Cattle Tick[J]. Elfos Scientiae, La Habana, Cuba, 1995, 200-204.
[89]. Lee,R.P, J.P Opdebeeck. Antigens identified by monoclonal antibodies in tissue sections of Boophilus microplus[J].Int.J.Parasitol, 1995, 25: 241-248.
[90]. Jonsson, N.N., Matschoss, A.L., Pepper, et al. Evaluation of tickGARD(PLUS), a novel vaccine against Boophilus microplus, in lactating Holstein–Friesian cows[J]. Vet. Parasitol, 2000, 88: 275–285.
[91]. Lina Mar?a Ruiz, Gemma Armengol, Edwin Habeych, et al. A theoretical analysis of codon adaptation index of the Boophilus microplus bm86 gene directed to the optimization of a DNA vaccine[J]. Journal of Theoretical Biology,2006, 239: 445–449.
[92]. Lina Marla Ruiz, Sergio Orduz, Elkin D. Lo′pez, et al. Immune response in mice and cattle after immunization with a Boophilus microplus DNA vaccine containing bm86 gene[J]. Veterinary Parasitology,2007, 144: 138–145.
[93]. Rodríguez M., Rubiera R., Penichet M., et al. High level expression of the B.micorplus Bm86 antigen in the yeast P.pastoris forming highly immunogenic particles for cattle[J]. Biotechnol. 1994, 33: 135-146.
[94]. Frisch J E,Towards a permanent solution for controlling cattle ticks [J]. Int. J. Parasito, 1991, 29: 57-71.
[95]. Pengally C,Antiparasitics. products and markets[M]. Animal Pharm Reports. PJB Publications. London: UK. 1999.
[96]. J. P. Dalton, Grace Mulcahy. Parasite vaccines—a reality [J]. Veterinary Parasitology, VOL98, July, 2001,149-167.
[2].孔繁瑶主编.家畜寄生虫学[M].北京:农业出版社,1981,293-296.
[3].邓国藩编著.中国经济昆虫志[M].北京:第十五册,蜱螨目蜱总科,科学出版社,1978,2.
[4].邓国藩,姜在阶主编.中国经济昆虫志[M].北京:第三十九册,蜱螨亚纲硬蜱科.科学出版社,1991,2.
[5]. Balashov,Y.S. Bloodsucking tick(Ixodoidea)-vector of disease of man and animals (English translation)[J].Misc.Publ.Entomol. Soc. Amer.1972,8:163-376.
[6].于心,叶瑞玉,龚正达.新疆蜱类志[M].乌鲁木齐:新疆科学技术卫生出版社.1997,79.
[7].阿布力克木·阿不都热西提,李冰,蒋卫等.吐鲁番地区首次发现小亚璃眼蜱的报告[J].地方病通报,2006,6 (21) : 10-11.
[8].罗建勋,殷宏,关贵全,等.小亚璃眼蜱对牛巴贝斯虫未定种和环形泰勒虫传播的试验研究[J].畜牧兽医学报,2005,36 (11):1197-1201.
[9]. Luo J X , Yin H , Guan G Q , et al. Description of new B abesia sp. infective for cattle in China [J]. Parasitology Research,2002,88 : S13–S15.
[10].罗建勋,殷宏,刘光远,关贵全,等.牛的巴贝斯虫18S rRNA基因序列比较研究[J].畜牧兽医学报,2005,36 (9):906-911.
[11].薄新文,汪明.宿主对蜱免疫的研究进展[J].中国兽医杂志,1998,24(10):45-47.
[12].袁改玲.青海血蜱生活史及不同发育阶段蛋白分析[D].呼和浩特,内蒙古农业大学,2002.
[13]. Wikel SK., Alarcon-Chaidez F.J. Progress toward molecular caracterization of ectoparasite moduoation of host immunity[J].Veterinary Parasitolog,2001,101: 275-287.
[14]. Benjamin L.HARI. Role of crooming in biological control of ticks[J]. Annals of the new academy of sciences, 2000,12:565-569.
[15].赵忠芳,孔繁瑶等.兔非特异性免疫力抗长角血蜱幼虫感染能力的研究[J].中国农业大学学报, 1998,(增刊):79-81.
[16].童竞亚主编.医学免疫学与微生物学[M].北京:人民卫生出版社,1993.
[17]. Johnston LAY, Kemp DH. Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks. Effects of induced immunity on tick populations [J]. International Journal for Parasitology,1986, 16: 27-34.
[18]. Kemp DH,Agbede RIS,Johnston LAY.Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: feeding and survival of the parasite on vaccinated cattle [J]. International Journal for Parasitology, 1986, 16: 115.
[19]. Opdebeeck J.P., Wong J.Y.M., Jackson L.A. Vaccines to protect Hereford cattle againstthe cattle tick, Boophilus microplus [J]. Immunology, 988, 63: 363.
[20]. Willadsen P, Riding GA, Mckenna RV. Immunologic Control of A Parasitic arthropod: Identification of a Protective Antigen from Boophilus microplus [J]. The Journal of Immunology, 1989, 143(4): 1346-1351.
[21]. Willadsen P, Eisemann CH, Tellam RL.“Concealed”antigens: expanding the range of immunological targets [J]. Parasitol Today, 1993, 9: 132.
[22]. Tsuda A, Mulenga A, Sugimoto C. cDNA cloning, characterization and vaccine effect analysis of Haemaphysalis longicornis tick saliva protein [J]. Vaccine, 2001, 19: 4287-4296.
[23]. Tsuji N, Kamio T, Isobe T. Molecular characterization of a peroxiredoxin from the hard tick Haemaphysalis longicornis[J]. Insect Molecular Biology, 2001, 10(2): 121-129.
[24]. Willadsen P., Riding G.A., Mckenna R.V., et al. Immunologic Control of A Parasitic Arthropod: Identification of a Protective Antigen from Boophilus microplus [J].The Journal of Immunology, 1989,143(4): 1346-1351.
[25]. Penichet M., Rodriguez M., Castellano O., et al. Detection of Bm86 antigen in different strains of Boophilus microplus and effectiveness of immunization with recombinant Bm86 [J]. Parasite Immunology, 1994, 16: 493-500.
[26]. Rand K.N., Moore T., Sriskantha A., et al. Cloning and expression of a protective antigen from the cattle tick Boophilus microplus [J]. Proc. Natl. Acad. Sci. USA. 1989, 86: 9657-9661.
[27]. Turnbull I.F., Smith D.R.J., Sharp P.J., et al. Expression and Secretion in Aspergillus nidulans and Aspergillus niger of a Cell Surface Glycoprotein from the Cattle Tick, Boophilus microplus, by Using the Fungal amdS Promoter System[J]. Applied and Environmental Microbiology, 1990, 56(9): 2847-2852.
[28]. Rodríguez M., Rubiera R., Penichet M., et al. High level expression of the B.micorplus Bm86 antigen in the yeast P.pastoris forming highly immunogenic particles for cattle [J]. Biotechnol, 1994, 33: 135-146.
[29]. Montesino R., Cremata J., Rodríguez M., et al. Biochemical characterization of the recombinant Boophilus microplus Bm86 antigen expressed by transformed[J]. Pichia pastoris cells. Biotechnol. Appl. Biochem, 1996, 23: 23-28.
[30]. García-García J.C., Montero C., Rodríguez M., et al. Effect of particulation on the immunogenic and protective properties of the recombinant Bm86 antigen expressed in Pichia pastoris [J]. Vaccine,1998, 16(4): 374-381.
[31]. García-García J.C., Montero C., Redondo M., et al. Control of ticks resistant to immunization with Bm86 in cattle vaccinated with the recombinant antigen Bm95 isolated from the cattle tick, Boophilus microplus[J]. Vaccine. 2000, 18: 2275-2287.
[32]. Riding G.A., Jarmey J., McKenna R.V., et al. A Protective“concealed”Antigen fromBoophilus microplus: Purification, Localization, and Possible Function [J]. The Journal of Immunology, 1994, 153: 5158-5166.
[33]. McKenna R.V., Riding G.A., Jarmey J.M., et al. Vaccination of cattle against the Boophilus microplus using a mucin-like membrane glycoprotein[J]. Parasite Immunology, 1998, 20: 325-336.
[34]. Rose R.D., McKenna R.V., Cobon G., et al. Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep[J]. Veterinary Immunology and Immunopathology, 1999, 71: 151-163.
[35]. Lee R.P., Opdebeeck J.P. Antigens Identified by Monoclonal Antibodies in Tissue Sections of Boophilus microplus [J]. International Journal for Parasitology, 1995, 25(2): 241-248.
[36]. Lee R.P., Opdebeeck J.P. Isolation of protective antigens from the gut of Boophilus microplus using monoclonal antibodies [J]. Immunology, 1991, 72: 121-126.
[37]. Toro-Ortiz R.D., et al. Monoclonal antibodies against Boophilus microplus and their effects on tick reproductive efficiency [J]. Veterinary Parasitology,1997, 69: 297-306.
[38].樊瑞泉,罗建勋,杨孝朴,殷宏.微小牛蜱Bm86基因的克隆与原核表达[J].甘肃农业大学学报,2007,42(1):15-19.
[39].李文卉,罗建勋,殷宏,等.微小牛蜱Bm86基因的真核表达[J].中国兽医科技,2005,35(9):718-722.
[40]. Mulenga A., Sugimoto C., Sako Y., et al. Molecular Characterization of a Haemaphysalis longicornis Tick Salivary Gland-Associated 29-Kilodalton Protein and Its Effect as a Vaccine against Tick Infestation in Rabbits[J]. Infection and Immunity,1999, 67(4): 1652-1658.
[41]. Tsuda A, Mulenga A, Sugimoto C, et al. cDNA cloning, characterization and vaccine effect analysis of Haemaphysalis longicornis tick saliva protein[J]. Vaccine, 2001, 19: 4287-4296.
[42]. You M., Xuan X., Tsuji N., et al. Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis[J]. Insect Biochemistry and Molecular Biology ,2001, 32: 67-73.
[43]. Tsuda A., Mulenga A., Sugimoto C., et al. cDNA cloning, characterization and vaccine effect analysis of Haemaphysalis longicornis tick saliva protein[J]. Vaccine, 2001, 19: 4287-4296.
[44]. Neurath H. The versatility of proteolytic enzymes. J Cell Bioche,1986, 32: 35-49
[45]. Rao M.B., Tanksale A.M., et al. Molecular and biotechnology aspects of microbial proteases [J]. Microb. Mol. Biol. Rev,1998, 62: 597-635.
[46]. Mulenga M., Sugino M., Nakajima M., et al. Tick-encoded serine proteinase inhibitors (serpins): Potential target antigens for tick vaccine development [J]. J. Vet. Med. Sci, 2001a, 63: 1063-1069.
[47]. Liao M, Zhou J, Hatta T, et al. Molecular characterization of Rhipicephalus(Boophilus) microplusBm86 homologue from Haemaphysalis longicornis ticks[J]. VetParasitol. 2007, 11(2): 146-157.
[48].高金亮.青海血蜱CDNA表达文库的构建和隐藏抗原的筛选、克隆与表达[D].北京：中国农业科院研究生院，2004.
[49].赵海平.青海血蜱幼蜱CDNA表达文库的构建及免疫学筛选[D].北京：中国农业科院研究生院，2007.
[50]. S. Ghosh N. K. Singh G. Das .Assessment of duration of immunity in crossbredcattle immunized with glycoproteins isolated from Hyalomma anatolicum anatolicumand Boophilus microplus[J].Parasitol Res, 2005, 95: 319–326.
[51]. Ghosh S, Khan MH. Isolation of immunoreactive proteins from the larval extracts ofBoophilus microplus and Hyalomma anatolicum anatolicum[J].Indian J Anim Sci,2000a, 70: 14–18.
[52]. Leboulle.G,Rochez C,Louahed J, et al. Ixodes ricinus salivary gland mRNAencoding factors induced during blood feeding[J].Am J Trop Med Hvg, 2002, 6(3):225-233.
[53]. Gill H S , Boid R , Ross C A. Isolation and characterization of salivary antigens fromHyalom ma anatolicum anatolicum [J]. ParasiteImmunology, 1986, 8: 11225.
[54]. Sran H.S.,Grewal A.S.,Kondal J.K. et al. Enhanced immunity to Hyalommaanatolicum anatolicum ticks in cross-bred calves using the tick salivary glandextractantigens[J].Veterinary Immunologyand Immunopathology, 1996, 51: 333-343.
[55]. Sharma,J.K.,Ghosh, et al Immunoprotective efficacy of a purified 39kDa nymphalantigen of Hyalomma anatolicum anatolicum[J]. Vaccine, 2001, 33(2): 103-116.
[56]. Kaba SA, Musoke AJ, Schaap D, et al. Novel baculovirus derived p67 subunitvaccines efficacious against East Coast fever in cattle[J]. Vaccine, 2005,23:2791–2800.
[57]. Musoke A, Rowlands J, Nene V, et al. Subunit vaccine based on the p67 majorsurface protein of Theileria parva sporozoites reduces severity of infection derivedfrom field tick challenge [J].Vaccine , 2005, 23: 3084–3095.
[58]. S. Ghosh P. Azhahianambi Jose, de la Fuente. Control of ticks of ruminants, withspecial emphasis on livestock farming systems in India: present and future possibilitiesfor integrated control—a review [J]. Exp Appl Acarol, 2006, 40: 49–66.
[59]. SANDER DE VOS1, LAURA ZEINSTRA1, OMAR TAOUFIK1, et al. Evidence forthe utility of the Bm86 antigen from Boophilus microplus in vaccination against othertick species [J]. Experimental and Applied Acarology , 2001,25: 245–261.
[60]. Mulenga A., Tsuda A., Onuma M., et al. Four serine proteinase inhibitors (serpin) fromthe brown ear tick, Rhiphicephalus appendiculatus: cDNA cloning and preliminarycharacterization[J]. Insect Biochemistry and Molecular Biology, 2003, 33: 267-276.
[61]. Kopacek P., Zdychova J., Yoshiga T., et al. Molecular cloning, expression and isolationof ferritins from two tick species- Ornithodoros moubata and Ixodes ricinus[J]. Insect Biochem Mol Biol, 2003, 33: 103-113.
[62].周金林.蜱的功能分子研究及其应用前景.动物医学进展[J]. 2004,52(1):53-56.
[63]. David Odongo, Lucy Kamau, Robert Skilton, et al. Vaccination of cattle with TickGARD induces cross-reactive antibodies binding to conserve Boophilus decoloratus[J]. Vaccine,2007,25: 1287–1296.
[64]. Almaza′n C, Kocan KM, Bergman DK, et al. Identification of protective antigens for the control of Ixodes scapularis infestations using cDNA expression library immunization [J]. Vaccine, 2003a, 21: 1492–1501.
[65]. Almaza′n C, Kocan KM, Bergman DK, et al. Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of expressed sequence tags [J]. Gene Ther Mol Biol , 2003b, 7: 43–59.
[66]. Almaza′n C, Blas-Machado U, Kocan KM, et al. Characterization of three Ixodes scapularis cDNAs protective against tick infestations [J].Vaccine,2005a,23: 4403–4416.
[67]. Almaza′n C, Kocan KM, Blouin EF, et al. Vaccination with recombinant tick antigens for the control of Ixodes scapularis adult infestations[J].Vaccine,2005b,23: 5294–5298.
[68]. de la Fuente J, Kocan KM . Strategies for development of vaccines for control of ixodid tick species [J]. Parasite Immunol, 2006, 28: 275–286.
[69]. Nijhof AM, Taoufik A, de la Fuente J, et al. Gene silencing of the tick protective antigens,Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference [J]. Int J Parasitol, 2006.10: 1015-1017.
[70]. Kocan KM.The tick protective antigen, 4D8, is a conserved protein involved in modulation of tick blood digestion and reproduction[J].Vaccine, 2006, 24: 4082–4095.
[71]. Katherine M. Kocan. Raúl Manzano-Roman,et al.Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA[J]. Parasitol Res, 2007, 100: 1411–1415.
[72]. Aljamali MN, Sauer JR, Essenberg RC. RNA interference applicability in tick research[J]. Exp Appl Acarol, 2002, 28: 89–96.
[73]. Almazán C, Kocan KM, Bergman DK, et al. Identification of protective antigens for the control of Ixodes scapularis infestations using cDNA expression library immunization[J]. Vaccine ,2003, 21: 1492–1501.
[74]. Almazán C,Blas-Machado U,Kocan KM,et al.Characterization of three Ixodes scapularis cDNAs protective against tick infestations[J].Vaccine, 2005,23: 4403–4416.
[75]. de la Fuente J, Kocan KM. Strategies for development of vaccines for control of ixodid tick species[J]. Parasite Immunol, 2006, 28: 275–283.
[76]. de la Fuente J, Almazán C, Blouin EF, et al. RNA interference screening in ticks for identification of protective antigens[J]. Parasitol Res, 2005, 96: 137–141.
[77]. de la Fuente J, Kocan KM . Advances in the identification and characterization of protective antigens for recombinant vaccines against tick infestations[J]. Expert Rev Vaccines, 2003, 2: 583–593.
[78]. de la Fuente J, Almazán C, Naranjo V, et al. Synergistic effect of silencing the expression tick protective antigens 4D8 and Rs86 in Rhipicephalus sanguineus by RNA interference[J]. Parasitol Res, 2006b, 99: 108–113.
[79]. de la Fuente J, Almazán C, Blouin EF, et al. Reduction of tick infections with Anaplasma marginale and A. phagocytophilum by targeting the tick protective antigen subolesin[J]. Parasitol Res, 2006d, 100: 85–91.
[80]. Taoufik Amar, Nijhof Ard M, de la Fuente, et al. Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference[J]. International Journal for Parasitology, 2006, 11: 5.
[81]. Willadsen P., Bird P., Cobon G.S., et al. Commercialisation of a recombinant vaccine against Boophilus microplus[J]. Parasitology, 1995, 110: S43-S50.
[82]. Tellam R.L., Smith D., et al. Vaccination against ticks[J]. In Animal Parasite Control Utilizing Biotechnology, 1992, 12: 303-331.
[83]. Rodriguez M., Penichet M.L., Mouris A.E., et al. Control of Boophilus mircoplus population in grazing cattle vaccinated with a recombinant Bm86 antigen preparation[J]. Vet parasitol,1995a, 57: 339-349.
[84]. Canales M., Enríquez A., Ramos E., et al. Large-scale production in Pichia Pastoris of the recombinant vaccine Gavac? against cattle tick[J]. Vaccine. 1997, 15(4): 414-422
[85]. Rodríguez M., Massard C.L., Fonseca A.H.D., et al. Effect of vaccination with a recombinant Bm86 antigen preparation on natural infestations of Boophilus microhlus in grazing dairy and beef pure and cross-breed cattle in Brazil[J]. Vaccine, 1995b, 13(18): 1804-1808.
[86]. Rodríguez M., Montero C., et al. Effect of vaccination with Gavac? on the incidence of Babesia bovis infections and the reduction in the number and frequency of acaricide treatments in cattle under production conditions in Cuba. In: Recombinant Vaccines for the Control of Cattle Tick[J]. Elfos Scientiae, La Habana, Cuba, 1995c, 187-194.
[87]. Vanegas L.F., Parra S.A., et al. Commercialization of the recombinant Vaccine Gavac? against Boophilus microplus in Colombia. In: Recombinant Vaccines for the Control of Cattle Tick[J]. Elfos Scientiae, La Habana, Cuba, 1995, 196-199.
[88]. Massard C.L., Fonseca R.N., et al. Effect of Vaccination with Gavac? on the reduction in the number and frequency of acaricide treatments in cattle under production conditions in Brazil. In: Recombinant Vaccines for the Control of Cattle Tick[J]. Elfos Scientiae, La Habana, Cuba, 1995, 200-204.
[89]. Lee,R.P, J.P Opdebeeck. Antigens identified by monoclonal antibodies in tissue sections of Boophilus microplus[J].Int.J.Parasitol, 1995, 25: 241-248.
[90]. Jonsson, N.N., Matschoss, A.L., Pepper, et al. Evaluation of tickGARD(PLUS), a novel vaccine against Boophilus microplus, in lactating Holstein–Friesian cows[J]. Vet. Parasitol, 2000, 88: 275–285.
[91]. Lina Mar?a Ruiz, Gemma Armengol, Edwin Habeych, et al. A theoretical analysis of codon adaptation index of the Boophilus microplus bm86 gene directed to the optimization of a DNA vaccine[J]. Journal of Theoretical Biology,2006, 239: 445–449.
[92]. Lina Marla Ruiz, Sergio Orduz, Elkin D. Lo′pez, et al. Immune response in mice and cattle after immunization with a Boophilus microplus DNA vaccine containing bm86 gene[J]. Veterinary Parasitology,2007, 144: 138–145.
[93]. Rodríguez M., Rubiera R., Penichet M., et al. High level expression of the B.micorplus Bm86 antigen in the yeast P.pastoris forming highly immunogenic particles for cattle[J]. Biotechnol. 1994, 33: 135-146.
[94]. Frisch J E,Towards a permanent solution for controlling cattle ticks [J]. Int. J. Parasito, 1991, 29: 57-71.
[95]. Pengally C,Antiparasitics. products and markets[M]. Animal Pharm Reports. PJB Publications. London: UK. 1999.
[96]. J. P. Dalton, Grace Mulcahy. Parasite vaccines—a reality [J]. Veterinary Parasitology, VOL98, July, 2001,149-167.