褐黄血蜱的鉴定与4D8基因的全长和表达分析

发布时间：2018-03-26 17:56

本文选题：褐黄血蜱　切入点：4D8　出处：《湖南农业大学》2015年硕士论文

【摘要】：目的：蜱以吸血为生,对人和动物有直接和间接的危害。本文以褐黄血蜱为对象,研究其功能基因4D8的全长序列,及测定4D8在不同阶段、不同组织的表达水平,为开发广谱抗蜱疫苗提供依据。方法：1、本实验借助体视显微镜对蜱样进行形态观察；PCR扩增COX1与ITSl序列；克隆测序PCR产物；分析所测序列同源性。2、比对已知血蜱属的4D8 ORF序列,设计引物扩增褐黄血蜱4D8 ORF序列；以此序列为基础设计引物扩增4D8 ORF的3’与5’端,获得4D8的全长序列；分析褐黄血蜱4D8 ORF与其它蜱种的同源性。3、对模板1、5、25、125、625倍稀释,建立目的基因4D8和管家基因β-actin的标准曲线；在最优条件下,RT-PCR测定4D8在褐黄血蜱不同阶段、不同组织的表达水平。结果：采自河南信阳、湖南华容刺猬,山西太谷犬体表的蜱均为褐黄血蜱；且其分布范围和宿主范围比已有的记载更大。褐黄血蜱4D8的全长为1318bp,开放性阅读框的大小为540bp,4D8 ORF序列在种属间高度保守。RT-PCR测定4D8表达量在蜱的不同阶段,雌蜱随其生长过程(从饱血若蜱、1/4饱血、半饱血到饱血)上调,饱血雌成蜱高于饱血雄成蜱；在不同组织,半饱血蜱明显高于饱血蜱。
[Abstract]:Objective: to study the full-length sequence of functional gene 4D8 and determine the expression level of 4D8 in different stages and tissues of Haemaphysalis brownicolor, which is a direct and indirect hazard to human beings and animals. In order to provide the basis for the development of broad-spectrum anti-tick vaccine, we used stereoscopic microscope to amplify the COX1 and ITSl sequences of tick samples, clone and sequence the PCR products, analyze the homology of the detected sequences, and compare the known 4D8 ORF sequences of Haemaphysalis. Primers were designed to amplify the 4D8 ORF sequence of Haemaphysalis brownicolor, and primers were designed to amplify the 3'and 5'ends of 4D8 ORF to obtain the full-length 4D8 sequence. The homology of 4D8 ORF and other ticks was analyzed. The template was diluted by 25125625 times. The standard curves of target gene 4D8 and housekeeping gene 尾 -actin were established, and the expression levels of 4D8 in different tissues of Haemaphysalis brownicolor at different stages were determined by RT-PCR under the optimal conditions. Results: Hedgehog sinensis was collected from Xinyang, Henan Province, Hunan Province. All the ticks on the surface of Taigu dog in Shanxi were Haemaphysalis brownicolor. The 4D8 of Haemaphysalis brownicolor was 1318 BP in length, and the size of open reading frame was 540 BP, 4D8 ORF sequence was highly conserved among species. RT-PCR was used to detect the expression of 4D8 in different stages of tick. Female ticks were up-regulated with the growth process (from 1 / 4 full blood, half full blood to full blood), and the female adult ticks were higher than male ones, and in different tissues, hemicyrifos were significantly higher than hemopyrifos.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.746

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