【作者】 赵金国；

【导师】 罗建勋； 曾巧英； 殷宏；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2008， 硕士

【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。更多还原

【Abstract】 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.更多还原