亚洲璃眼蜱唾液腺功能基因的筛选和鉴定

The different expression genes in Hyalomma asiaticum salivary glands were investigated by cDNA microarray analysis. The clones were selected from the cDNA libraries constructed using the technique of suppression subtractive hybridization (SSH). Insert fragments of SSH cDNA libraries were amplified by PCR, and the PCR products were then purified and robotically printed onto aminosilane-coated glass slides, and each clone was spotted in the same slides. The cDNA arrays were hybridized with the Cy3-dUTP and Cy5-dUTP probes from unfed and engorged tick cDNA, respectively. A dye-swap experiment was performed with that of cDNA microarray. The hybridization signal of each spot was measured, normalized and analytical as a Ratio. The complete different expression gene was obtained by RACE approach. The Open Reading Frame (ORF) of the gene ware cloned into a FastBac vector, and was transformed into a E. coll DH10Bac cells. Recombinant Bacmid DNA (rBacmid) was obtained and used to transfect insect cells SF9. The recombinant protein products were assessed for qualitative and quantitative analysis by SDS-PAGE and western-blots that the products were separated by the AKTA Purifier FPLC system. the effects of the recombinant protein on TT, PT, APTT, the attachment, the shape and composition of the cement cone are observed. the rabbit were immunized with recombinant protein and then were infested with the H.asiaticum, in order to evaluate the immunogenicity and specific-reactivity of the recombinant protein. Furthermore, its affection on tick blood-sucking behavior, blood period (days), blood-meal (mg) and attachment. were observed.

将亚洲璃眼蜱唾液腺SSH cDNA文库各片段的PCR 产物点样于经处理的基片上，制成cDNA 芯片；以Cy3-dUTP和Cy5-dUTP将不吸血蜱和吸血蜱mRNA反转录成cDNA探针；与预制好的cDNA芯片杂交；分析、处理各点信号，计算Ratio值，挑出差异表达基因(片段)。 RACE法全长差异表达基因。将ORF克隆到FastBac系列表达载体中，转化DH10Bac感受态细胞，制备重组杆状病毒DNA(rBacmid)，转染sf9昆虫细胞。借助SDS-PAGE、Western-Blots对重组蛋白进行定性分析和定量分析。用AKTA Purifier快速液相色谱仪(FPLC)分离、纯化重组蛋白。 体外实验观察重组蛋白对TT、PT，APTT，粘合结形态和成分的影响。免疫家兔，测定重组蛋白的免疫原性和特异反应性；进行饲蜱试验，考查蜱在吸血行为、饱血时间、饱血均量、粘合结节等方面的变化。

褐黄血蜱广泛分布于我国西北地区以外的各省、市、自治区，侵袭多种哺乳动物和鸟类，携带新布尼亚病毒、立克氏体和包氏螺旋体等多种病原，具有潜在的公共卫生意义。.联系生产实际，更新技术方法，以褐黄血蜱为对象，以LC/MS/MS和NGS等技术，全面、系统地探究了蜱类功能基因和活性蛋白，取得的成效如下：.⑴采用LC/MS2法分析褐黄血蜱唾液、粪便和中肠内容物的蛋白成分，从中鉴定高可信度蛋白52种、21种和15种。运用iTraq等方法测定饱血和半饱血两状态下雌成蜱唾液和中肠内容物中的各蛋白含量，找出差异表达蛋白51种和80种；.⑵采用NGS测序技术进行褐黄血蜱唾液腺和中肠转录组学分析。由饱血和半饱血褐黄血蜱唾液腺和中肠检出的reads聚类生成Unigenes 分别为54357个和76556个，其中差异表达者5295个和5508个；.⑶Mascot法综合分析蛋白组学和转录组学数据，搜获有益Unigene 441个，其中111个注释为高可信度蛋白。以此为据设计引物，已克隆出褐黄血蜱唾液、中肠内容物和粪便中高丰度存在的Enolase和HSC70等16个差异表达的高度可信蛋白cDNA；.⑷采用RT-PCR技术分析Enolase、Subolesin和HSC70等9种基因在不同状态下的表达水平；.⑸借助真/原核表达体系制备Enolase、haemalin、AV422、Serpin-2和HSP70-5等5种蛋白的生物活性。研究结果表明，HSC70在蜱血餐抗凝中发挥重要作用，课题组也将该蛋白确定为今后研究的重点；.⑹运用PCR-DGGE和illumina两种手段分析了褐黄血蜱的唾液、中肠内容物的菌群结构，对其传病风险进行评估；.⑺根据实施本项目获得的数据、撰写论文27篇，已发表24篇(SCI收录14篇)、录用待发2篇；.⑻获批国家专利1项；.⑼获中国农业科学院国家重点实验室开放基金课题1项；⑽培养博士生2人，硕士生12人。