【作者】 沈辉；

【导师】 刘光远；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2012， 硕士

【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。更多还原

【Abstract】 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.更多还原