【作者】 田占成；

【导师】 殷宏； 刘光远； 罗建勋；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2010， 博士

【摘要】 蜱可传播人和动物的多种疾病病原,特别是森林脑炎、新疆出血热、莱姆病等人畜共患病,是最重要的人畜共患病传播媒介之一,严重威胁着人类健康和畜牧业生产。蜱的防治成为畜牧业生产中迫切需要解决的问题。目前采用的化药杀虫剂防治蜱的传统方法存在蜱耐药性和环境污染的缺陷,因此研发新的防控制剂成为必然趋势。本研究拟采用免疫学方法和cDNA末端快速扩增技术,克隆、表达长角血蜱(Haemaphysalis longicornis)和青海血蜱(H. qinghaiensis)的与繁殖发育相关的功能基因,为探索蜱生理行为分子调控机制、评价新型环境友好害虫控制制剂和抗蜱疫苗候选抗原提供平台,填补我国在蜱生理行为干预及防控新制剂研究方面的空白,为我国蜱的防控另辟一条新途径。主要研究结果如下：1)通过免疫学筛选的方法和RACE技术,在已构建的长角血蜱卵cDNA表达文库和青海血蜱中克隆获得了20个有效表达序列标签(EST),目前获得了其中12个基因的全长序列。为进一步解析硬蜱基因编码蛋白具体的生理作用机制奠定了物质基础,为研制抗蜱疫苗建立了候选抗原的平台。2)从长角血蜱卵cDNA文库获得了核糖体蛋白L24的部分序列,通过cDNA末端快速扩增技术(RACE)获得了该基因的全长序列。实时定量RT-PCR分析表明,该基因在长角血蜱的各个组织和发育阶段均表达；在部分饱血雌蜱中,与其他组织相比,L24主要在卵巢和唾液腺中表达,卵和幼蜱发育阶段的核糖体蛋白L24表达水平明显低于其他发育阶段。从青海血蜱和小亚璃眼蜱(Hyalomma anatolicum anatolicum)中也分别克隆到了核糖体蛋白L24基因,氨基酸序列比较表明,其同源性较高,只有几个氨基酸发生了变异。HLL24重组蛋白免疫兔体保护性试验结果表明,免疫组与对照组相比,长角血蜱雌蜱各项生理指标下降不显著。3)从长角血蜱卵cDNA表达文库中筛选到了一个长角血蜱热休克蛋白类似物—Hsp70基因。Hsp70基因全长为2311bp,完整阅读框长度为1983bp,编码661个氨基酸,预测分子量为72.5 kDa,等电点为5.2。具有Hsp70家族中高保守功能修饰域和内质网定位蛋白中普遍存在的内质网延伸信号"KDEL"。Hsp70与肩突硬蜱(Ixodes scapularis)Hsp70的氨基酸序列相似性达90%,与原鸡(Gallus gallus)的78kDa葡萄糖调控蛋白前体(78 kDa glucose-regulated protein precursor)相似性为85%。实时定量RT-PCR分析表明：部分饱血的长角血蜱雌蜱的卵巢和唾液腺中的Hsp70表达水平明显高于其他组织。尽管Hsp70的表达水平在饥饿长角血.蜱不同发育阶段表达水平很低,但通过吸血可明显诱导其表达水平的增加。Hsp70的表达水平与温度的变化相一致,表现出了明显的冷热应激典型特征(4℃-37℃)。免疫印迹分析表明：重组长角血蜱Hsp70蛋白免疫兔体制备的抗体与卵粗抗原的100 kDa、72.5 kDa和28.4 kDa的3条天然蛋白条带产生明显反应,但在部分饱血蜱的蛋白抽提物中只检测到一条72.5 kDa的天然蛋白带与之反应。Hsp70重组蛋白的免疫保护性实验表明,该蛋白对长角血蜱雌蜱的饱血程度和产卵率的下降作用影响不明显。综上所述,尽管Hsp70的表达水平可明显通过吸血的生理行为来诱导上调,但定位于内质网的长角血蜱Hsp70作为组成型表达基因可能不适合于用作抗蜱疫苗候选抗原。4)采用cDNA末端快速扩增技术(RACE)克隆了青海血蜱含rabGAP域蛋白(TBC1D13)的全长序列。TBC1D13基因全长为1702bp,编码396个氨基酸,预测分子量为46.09 kDa,等电点为6.0。通过建立的各物种TBC1D13发育遗传树和氨基酸序列比对结果表明：TBC1D13基因的发育遗传进化保守,蜱源TBC1D13与脊椎动物来源TBC1D13形成一个发育分支,远离于昆虫TBC1D13的发育遗传分支。实时定量RT-PCR分析表明：TBC1D13在青海血蜱雌蜱吸血期间,其于唾液腺中的表达水平明显高于其他组织。免疫印迹分析表明,在青海血蜱唾液腺中,天然TBC1D13蛋白的分子量约为49 kDa。重组TBC1D13蛋白免疫兔体诱导了强的免疫反应,产生了较高的抗体滴度。TBC1D13重组蛋白免疫兔体保护性试验结果表明：在统计学上,与GST蛋白免疫兔体对照组相比,攻击的雌蜱饱血程度和产卵重量都有明显下降,但该重组蛋白对宿主动物可能产生一定的副作用。因此,尽管TBC1D13基因对青海血蜱的存活机制很关键,但是否可作为疫苗候选抗原对蜱进行防控,其生物安全性还需要在兔体模型和其他实验动物模型上进行进一步的评估。更多还原

【Abstract】 Many zoonosis such as Forest Encephalitis, Xinjiang Hemorrhagic Fever and Lyme disease caused by pathogens transmitted by the hard ticks, which seriously threat the health of mankind and the development of husbandry. The control of ticks is emergent question needed to be solved. Currently the principal tick control method in China is the application of acaricides. This approach is, however, associated with a number of disadvantages such as chemical pollution of the food chain and the environment as well as the potential for development of resistance against acaricides by ticks.it is necessary to develop new tick control agent. The study cloned and expressed the gene involed in the development and propagation from Haemaphysalis longicornis and H. qinghaiensis ticks by the method of immunoscreening and cDNA end rapid amplification (RACE). To offer the platform for investigating the molecular mechanism of controlling on the physiological behavior of ticks and evaluating the neotype environmentally friendly pest biological agents and furthermore make up the deficiency in the intervention of physiological behavior and the controlling neotype agent. To develop a new channel for controlling ticks. The mostly results as followed:1) Twenty expression sequence tag (EST) were cloned from the cDNA expression library of H. longicornis egg and H. qinghaiensis by the method of immunoscreening and RACE. Among them, the complete sequence of 12 genes were obtained by the method of cDNA end rapid amplification, which offered the base for investigating the molecular mechanism of physiological behavior of ticks and the screening of the vaccine candidated antigen.2) A fragment of ribosomal protein L24 was obtained from the complementary deoxyribonucleic acid (cDNA) library of H. longicornis eggs. The complete sequence of the clone was subsequently obtained using rapid amplification of the cDNA ends (RACE). Ribosomal protein L24 from H. longicornis had a high percentage similarity to this protein from different species. Conserved domains were also identified in RpL24. Real-time polymerase chain reaction (PCR) analysis showed that this gene is expressed in various tissues and different developmental stages of H. longicornis. Furthermore, HLL24 is mostly expressed in ovaries and salivary glands compared with other tissues in partially fed adult female ticks, and the expression level of HLL24 is significantly lower in eggs and larvae than in other developmental stages. RpL24 was also cloned from H. qinghaiensis and Hyalomma anatolicum anatolicum ticks, respectively. Comparison of their amino acid sequences revealed difference only in several amino acids. A vaccine based on the HLL24 recombinant protein could not protect rabbits against H. longicornis.3) Heat shock protein 70 (Hsp70) was identified from cDNA library of H. longicornis eggs. The Hsp70 cDNA contains 2311 bp that encode 661 amino acid residues with the predicted molecular weight of 72.5 kDa and an isoelectronic point (pI) of 5.2. It also contains the highly conserved functional motifs of the Hsp70 family and a specific endoplasmatic reticulum retention signal "KDEL" that is common among ER-localized proteins. The Hsp70 exhibits 90% identity to the putative Hsp70 of Ixodes scapularis,85% to Gallus gallus 78 kDa glucose-regulated protein precursor. Real time RT-PCR analysis showed that the expression levels of the Hsp70 in ovaries and salivary glands were siginificantly higher than other tested tissues in partially fed adult female ticks. Although the expression level of the Hsp70 was constantly low in unfed ticks, it was significantly induced by blood-feeding. However, the expression was positively coordinated to the temperature (4℃-37℃, tested). Western Blots analysis showed that the rabbit antiserum against the recombinant H. longicornis Hsp70 protein recognized an about 100 kDa, an about 72.5 kDa, an about 28.4 kDa native protein in egg lysates and 72.5 kDa band was also detected in the protein extracts of partially fed larvae. Immunization using the Hsp70 recombinant protein did not result in the statistically significant reduction of female engorgement and oviposition. These results suggested that although HLHsp70 maybe played a certain role in the physiological activities of ticks, the HLHsp70 as a constitutive protein was not suitable to be selected as vaccine candidate antigen against ticks.4) H. qinghaiensis rabGAP-domain containing protein (TBC1D13) was cloned using rapid amplification of the cDNA ends (RACE). The TBC1D13 cDNA contains 1702 bp that encode 396 amino acid residues with a predicted molecular weight of 46.09 kDa and an isoelectronic point (pI) of 6.0. Phylogenetic analysis of the TBC1D13 from H. qinghaiensis compared with other members of TBC1 domian family 13 members available in GenBank showed that TBC1D13s were evolutionaily conserved and were greatly distant between vertebrate and invertebrate. Real time RT-PCR analysis showed that the expression level of the HqTBC1D13 was significant higher in salivary glands of partially fed females than in other tissues. Western Blot analysis indicated that the rabbit antiserum against the rTBC1D13 recognized an about 49 kDa native protein in salivary glands of partially fed females. Rabbit model vaccinated by rTBC1D13 produced higher antibody titer. Immunization using the recombinant TBC1D13 protein based on the rabbit model resulted in the statistically significant reduction of female engorgement and oviposition. However, it also maybe hed the adverse effect on the rabbit model compared with the control group. So whether the rHqTBC1D13 can be selected as vaccine against ticks is worthwhile to be further evaluated in the other animal model and rabbit model, although the HqTBC1D13 appeared to be important for the survival of ticks.更多还原