本文選題：褐黃血蜱　切入點：4D8　出處：《湖南農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：目的：蜱以吸血為生,對人和動物有直接和間接的危害。本文以褐黃血蜱為對象,研究其功能基因4D8的全長序列,及測定4D8在不同階段、不同組織的表達(dá)水平,為開發(fā)廣譜抗蜱疫苗提供依據(jù)。方法：1、本實驗借助體視顯微鏡對蜱樣進(jìn)行形態(tài)觀察；PCR擴(kuò)增COX1與ITSl序列；克隆測序PCR產(chǎn)物；分析所測序列同源性。2、比對已知血蜱屬的4D8 ORF序列,設(shè)計引物擴(kuò)增褐黃血蜱4D8 ORF序列；以此序列為基礎(chǔ)設(shè)計引物擴(kuò)增4D8 ORF的3’與5’端,獲得4D8的全長序列；分析褐黃血蜱4D8 ORF與其它蜱種的同源性。3、對模板1、5、25、125、625倍稀釋,建立目的基因4D8和管家基因β-actin的標(biāo)準(zhǔn)曲線；在最優(yōu)條件下,RT-PCR測定4D8在褐黃血蜱不同階段、不同組織的表達(dá)水平。結(jié)果：采自河南信陽、湖南華容刺猬,山西太谷犬體表的蜱均為褐黃血蜱；且其分布范圍和宿主范圍比已有的記載更大。褐黃血蜱4D8的全長為1318bp,開放性閱讀框的大小為540bp,4D8 ORF序列在種屬間高度保守。RT-PCR測定4D8表達(dá)量在蜱的不同階段,雌蜱隨其生長過程(從飽血若蜱、1/4飽血、半飽血到飽血)上調(diào),飽血雌成蜱高于飽血雄成蜱；在不同組織,半飽血蜱明顯高于飽血蜱。
[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.
【學(xué)位授予單位】：湖南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.746

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本文編號：1668933