【作者】 张丽艳；

【导师】 刘光远；

【作者基本信息】 中国农业科学院 ， 预防兽医， 2010， 硕士

【摘要】 长角血蜱(Haemaphysalis longicornis)作为一种吸血性外寄生虫,是人和动物多种病原的重要传播媒介,可传播包括细菌、病毒、寄生虫、立克次氏体等引起的多种疫病,给我国畜牧业的发展带来严重威胁。目前对蜱的防控方法主要是化学药物防治,但随着蜱耐药性的产生以及药物残留、环境污染等公共卫生问题的出现,迫使人们寻求新的更为安全的防治措施。近年来免疫学防治方法成为人们的研究热点。而蜱功能基因的筛选、克隆及表达是免疫学防治的前提。本实验用兔抗长角血蜱雌性成蜱阳性血清、兔抗长角血蜱幼蜱阳性血清、兔抗长角血蜱卵阳性血清对已构建好的长角血蜱饥饿雌蜱全蜱cDNA表达文库进行免疫学筛选,共获得11个有用的cDNA序列,依次编号为HL01-HL11。核酸序列以及氨基酸序列分析显示HL07与长角血蜱卵黄蛋白原的相似性高达99%,其余均为长角血蜱新基因,编码蛋白与青海血蜱Hq05、青海血蜱肌原蛋白T、肌球蛋白碱性轻链分子、微小牛蜱副肌球蛋白、肩突硬蜱表皮蛋白以及多种蜱的线粒体蛋白等具有相似性。目前已有5个序列登录到GenBank上,登陆号依次为:GU932666、GU932667、GU932668、GU932669、GU932670。选用大肠杆菌BL21(DE3)pLysE作为表达菌株,对已获得的8个阳性克隆(HL02、HL04、HL05、HL06、HL07、HL09、HL10、HL11)的重组质粒进行原核表达。结果显示,除HL10外,其他基因均表达。分别以兔抗长角血蜱雌性成蜱阳性血清、兔抗长角血蜱幼蜱阳性血清为一抗,利用Western-blot方法检测这些重组蛋白的反应性,结果发现HL02、HL04、HL05、HL06均表现出良好的反应原性。这为抗蜱疫苗的研究提供了更多的候选基因。采用5’RACE技术成功获得了HL05 5’端未知序列,拼接后获得其全长。该基因全长1053bp,ORF含540个碱基,预测编码蛋白大小为20.01ku。生物信息学分析显示,该基因具备一个糖基化位点,有信号肽,属于分泌型蛋白,无跨膜结构,抗原指数高。将该基因全序列提交至GenBank,获得登录号为GQ499841。将重组质粒pSCREEN/HL05在大肠杆菌中成功进行大量表达,利用亲和层析法纯化重组蛋白,免疫动物,间接ELISA法检测抗体水平表明免疫效果良好,Western-blot方法检测蜱各阶段天然蛋白HL05的存在情况,结果显示该蛋白在幼蜱、若蜱、成蜱中均有表达,在卵中不存在。天然蛋白HL05可能具有3个亚型,大小分别是21ku、23 ku、55 ku。攻蜱实验表明该蛋白能够降低雌蜱的饱血体重,说明该基因能够诱导动物产生一定的抗蜱保护效应,可以作为抗蜱疫苗进行深入的研究。更多还原

【Abstract】 Haemaphysalis longicornis are blood sucking ectoparasites and served as one of the most important vectors transmitting bacteria, viruses, parasites and rickettsia. It has been posing a serious threat to the animal husbandry in China. At present, although the principal tick control measure is chemical acaricides method, the development of alternative methods for tick control is important and urgent due to the appearance of acaricide-resistant ticks, environmental and food chain contamination. The use of tick vaccines has been shown to be the most promising alternative tick control method. The success of this method depends on the identification, cloning and in vitro expression of tick molecules which play a key physiological role in the survival of ticks and spread of disease.In this study, a cDNA expression library of H. longicornis unfed female tick was screened with anti-H. longicornis unfed female tick serum, anti-H. longicornis larva serum and anti-H. longicornis egg serum produced from rabbit, respectively. Eleven genes encoding immunodominant antigens were obtained, and named for HL01 to HL11. The nucleotide and deduced amino acid sequence analysis showed that HL07 shared 99% similarity with H. longicornis vitellogenin, and the others including were novel gene of H. longicornis, which had significant homology with the H. qinghaiensis Hq05 gene, H. qinghaiensis myogenic protein T, H. qinghaiensis myosin alkali light chain molecules, Boophylus microplus paramyosin, Ixodes scapulars surface protein and mitochondria of a variety of tick, respectively. Five genes were submitted to GenBank, accession numbers were GU932666, GU932667, GU932668, GU932669, GU932670, respectively. Eight genes including HL02, HL04, HL05, HL06, HL07, HL09, HL10, HL11 were expressed in the Escherichia coli BL21(DE3)pLysE. All of them except HL10 were expressed successfully. The expression products of HL02, HL04, HL05 and HL06 gene revealed good reactogenicity by Western-blot with anti-H. longicornis unfed female tick serum and rabbit anti-H. longicornis larva serum. These will provide candidates for further screening of protective antigen genes of H. longicornis.The HL05 gene was amplified by 5’rapid amplification of cDNA ends (5’RACE), and the full-length cDNA was obtained by splicing. The full-length of HL05 gene was 1053bp and contained an open reading frame (ORF) of 540 bp that codes for 179 amino acid residues with a coding capacity of 20.01ku. Bioinformatics analysis indicated that HL05 protein has a glycosylation site, it was secreted, no transmembrane domain and more potential antigenic determinants. The full-length sequence was submitted to GenBank and accession number was GQ499841. The recombinant plasmid pSCREEN/HL05 was expressed in E. coli BL21 (DE3), and expression products were 55 ku. Affinity chromatography was used to purify the recombinant protein, and the protein HL05 had good immunogenicity by indirect ELISA. Native HL05 was detected in ticks larvae, nymphs and adult except egg successfully, and it may have two isoforms which were 21ku, 23 ku. Vaccination of rabbit with rHL05 resulted in reduction of engorged weight of female ticks compared to the controls, and it conferred that rHL05 is a significant protective immunity. These results revealed that rHL05 could be a candidate vaccine molecule for the control of H. longicornis.更多还原