【作者】 张维谊；

【导师】 黄维义； 周金林；

【作者基本信息】 广西大学 ， 预防兽医学， 2004， 硕士

【摘要】 镰形扇头蜱可传播牛的巴贝斯虫病等，是我国南方的优势蜱种。其唾液腺是重要器官，在吸血过程中分泌大量的功能分子，这些功能分子成为最有价值的候选疫苗抗原分子。本文通过对镰形扇头蜱唾液腺及其表达序列标签(EST)研究，旨在寻找抗蜱及蜱传病疫苗的抗原候选分子。 本研究选择温度为25℃、相对湿度为92％的条件进行镰形扇头蜱兔体的人工饲养和繁殖，获得单雌蜱克隆蜱群。解剖镰形扇头蜱，取其唾液腺，经液氮反复冻融、超声、离心获得唾液腺提取物(SGE)。将含量为10μg的镰形扇头蜱未吸血和半饱血SGE进行SDS-PAGE，发现二者蛋白表达存在明显差异，主蛋白条带分别为69kDa、100kDa，吸血后蛋白表达发生变化，在分子量为45-100kDa间产生一些新的蛋白分子。将蛋白抗原转印到PVDF膜，用被镰形扇头蜱三次叮咬的兔抗血清进行Western-blotting分析，发现35kDa的蛋白可能是较重要的抗原，100kDa蛋白是蜱吸血时产生的新抗原。用半饱血SGE抗原经间接ELISA法检测人工感染的镰形扇头蜱幼虫、若虫、成虫的免抗蜱唾液抗体的变化情况，发现被蜱叮咬的兔抗体效价随着叮咬次数的增加而增加。通过Smart方法结合长链PCR(LD-PCR)构建了高质量的镰形扇头蜱半饱血唾液腺cDNA广西大学硕士论文镰形扇头蝉唾液腺及其表达序列标签的研究文库，文库重组率为95.2%，插入片段多在0.5一Zkb之间，库容量为2、75X106pfu/mL，为分离有价值的基因提供了资源及平台。经大肠杆菌BM25.8质粒化，从eDNA文库获得1 00条平均长度为745bp的有效ESTs，经功能分析发现，分别与能量代谢、信号传导、蛋白合成等基因相关，Es丁s中有27%的未知新基因，2条分别为ATP酶、细胞色素氧化酶m蛋白的全长基因，并登录在Ge心ankTM上，登录号分别为AY319968、AY422796;同时在ESTs中还发现一些抑制分子、粘合素蛋白等有价值的ESTs。 通过研究，发现了镰形扇头蜂唾液腺的重要蛋白抗原;构建了国内第一个镰形扇头蟀唾液腺cDNA文库，并运用EST技术获得了新基因，为蟀的相关研究提供了平台，为控制蟀及蟀传病奠定了基础。更多还原

【Abstract】 Rhipicephalus haemaphysaloides haemaphysaloides is one of the most important ticks in South China and can cause many diseases such as bovine babesiasis. Its salivary gland is an important organ during biting and secretes a number of functional molecules which are potential candidates for vaccine development. The objectives of the present study were to examine the salivary gland’s compositions and to look for molecules which could be used vaccine candidates for controlling tick and tick-borne diseases.At 25℃ and relative humidity of 92%, R. haemaphysaloides haemaphysaloides was raised by biting rabbits and propagating. The salivary gland was obtained by dissecting the R. haemaphysaloides haemaphysaloides. Salivary gland extract (SGE) was extracted by freezing and thawing the salivary gland in liquid nitrogen several times, followed by homogenization by ultrasound and centrifugation.Ten micrograms of SGE from non-biting Rhaemaphysaloides haemaphysaloides and from those bitted for 5 days were analysed by SDS-PAGE, and the results revealed that their protein expressions were obviously different. The SGE changed after biting and produced some new molecules in the range of 45-100kDa. The proteins were transferred to PVDF and analysed by Western-blotting using the serum from rabbit which was bitted by R. haemaphysaloides haemaphysaloides thress times. The results indicated that the protein of 35 kDa could be a very important one and the protein of 100kDa is a newly expressed protein after biting. The dynamics of antibody responses in rabbit infected with the tick was examined using indirect ELISA. The antibody titer increased after biting and the more biting the higher titer.Using the ’Smart technique’ and LD-PCR, a cDNA library of the salivary gland of R. haemaphysaloides haemaphysaloides was constructed with a capacity of 2.75x106pfu/mL and a recombinant rate of 95%. The inserted fragments were in the range of 0.5-2.0kb. The constructed library provided the resources for cloning valuable new genes. 100 ESTs from this library with an average length of 745bp were obtained by random sequencing. 27% of the ESTs represent new unknown genes. The others included two complete genes, some inhibition factors, cements and other valuable ESTs. The results of the present studies have important implications for further studies of the salivary gland and for the development of molecular vaccines for controlling tick and tick-borne diseases.更多还原