青海血蜱AQPs4种转录异构体的克隆与蛋白结构分析

发布时间：2018-02-22 03:32

本文关键词： 青海血蜱水通道蛋白(AQPs) 生物信息学分析转录异构体无细胞表达　出处：《甘肃农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：蜱是专性吸血的外寄生虫,是传播多种人和动物疾病的第二大生物媒介,给人类健康和畜牧业带来极大危害。蜱的防控是近年国内外关注的焦点和研究热点。蜱对化学药物存在抗药性和环境污染等缺陷,新型抗蜱制剂具有广泛的应用前景。青海血蜱是仅在我国分布并传播多种病原,对畜牧业造成严重危害的蜱种之一。蜱叮咬宿主传播病原的过程中存在大量液体运输活动。水通道蛋白(AQPs)是广泛存在生物体细胞膜上一类专性介导液体跨膜转运的重要功能蛋白,已证明在液体运输中发挥关键作用,可能作为研究蜱疫苗的关键蛋白之一,但蜱体内AQPs研究甚少,生物学功能也不清楚。本研究根据NCBI数据库公布的6种蜱AQPs的基因序列设计兼并引物,以青海血蜱cDNA为模板,利用RT-PCR(reverse transcription polymerase chain reaction)方法,扩增获得青海血蜱AQPs的部分序列;在此基础上设计5’和3’RACE(rapid-amplification of cDNA ends)引物,运用RT-PCR和RACE方法扩增青海血蜱AQPs全长序列,经分析获得青海血蜱AQPs 4种转录异构体。生物信息学分析显示,获得的青海血蜱AQPs 4种转录异构体的氨基酸顺序只在3,末端存在差异,其余部分氨基酸顺序一致,均有6个跨膜区,无信号肽序列,疏水性较强;进化分析显示青海血蜱AQPs 4种转录异构体与Rhipicephalus appendiculatus,Rhipicephalus sanguineus的AQPs序列同源性较高(82%),初步说明获得的青海血蜱AQPs 4种转录异构体是正确的。根据上述获得的青海血蜱AQPs 4种转录异构体的基因序列,设计引物扩增保守区。将目的片段连接表达载体pET-28a(+),经PCR、双酶切及测序验证正确后,体外无细胞进行表达并纯化,SDS-PAGE及Western-blot方法验证了蛋白的正确表达;同时将AQPs蛋白的3个外模区域合成多肽,并用多肽制备相应的多克隆抗体。以上研究结果显示,青海血蜱AQPs基因存在转录异构体,对转录异构体相同片段进行无细胞表达并纯化,并利用合成的多肽制备相应多克隆抗体,为新型抗蜱药物或疫苗的研制提供了一定的基础,对揭示蜱体内液体运输机制和蜱防治具有重要意义。
[Abstract]:Ticks are specialized blood sucking parasites and the second largest biological vector for the spread of many human and animal diseases. In recent years, the control of ticks is the focus of attention and research focus at home and abroad. Ticks have the defects of resistance to chemical drugs and environmental pollution. The new anti-tick preparation has a wide application prospect. Haemaphysalis qinghaiensis is only distributed and spread a variety of pathogens in China. One of the ticks that cause serious harm to animal husbandry. There is a large amount of liquid transportation in the course of biting the host to spread the pathogen. Aquaporin aquaporins (AQPs) is a kind of specific liquid transmembrane transport on the cell membrane of organisms. Important functional proteins, It has been proved that it plays a key role in liquid transportation and may be one of the key proteins in the study of ticks vaccine, but there is little research on AQPs in ticks. In this study, degenerate primers were designed according to the gene sequences of six species of ticks AQPs published in NCBI database. Using RT-PCR(reverse transcription polymerase chain reactionation method, partial sequences of Haemaphysalis qinghaiensis AQPs were amplified by RT-PCR(reverse transcription polymerase chain reactionation method. On this basis, 5 'and 3 RACEC rapid-amplification of cDNA endings primers were designed to amplify the full-length AQPs sequence of Haemaphysalis qinghaiensis by using RT-PCR and RACE methods. Four transcriptional isomers of Haemaphysalis qinghaiensis AQPs were obtained by analysis and bioinformatics analysis. The amino acid sequence of the four transcriptional isomers of Haemaphysalis qinghaiensis was only 3, with the same amino acid sequence in the other parts, six transmembrane regions, no signal peptide sequence and strong hydrophobicity. Phylogenetic analysis showed that the four transcriptional isomers of Haemaphysalis qinghaiensis AQPs had high homology with the AQPs sequence of Rhipicephalus appendiculatusus Rhipicephalus sanguineus, which indicated that the four transcriptional isomers of Haemaphysalis qinghaiensis AQPs were correct. According to the above obtained AQPs, four AQPs species of Haemaphysalis qinghaiensis were obtained. The gene sequence of the transcriptional isomer, The primers were designed to amplify the conserved region. The target fragment was ligated into the expression vector pET-28a (pET-28a), which was confirmed by PCR-, double-enzyme digestion and sequencing, and then expressed without cells in vitro and purified by SDS-PAGE and Western-blot methods to verify the correct expression of the protein. At the same time, the polypeptides were synthesized from the three outer model regions of AQPs protein, and the corresponding polyclonal antibodies were prepared by the polypeptides. The results showed that the AQPs gene of Haemaphysalis qinghaiensis had transcriptional isomers. The same fragment of the transcriptional isomer was expressed and purified without cells, and the polyclonal antibody was prepared by using the synthesized polypeptide, which provided a basis for the development of new anti-tick drugs or vaccines. It is of great significance to reveal the mechanism of liquid transport in ticks and to control ticks.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.746

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