【摘要】：鈍刺血蜱(Haemaphysalis doenitzi)主要分布于中國的福建、云南、臺灣等地,多寄生在鳥類和小型哺乳動物體表上,能攜帶巴貝斯蟲等多種病原體,對野生動物和人類造成嚴(yán)重危害。防御素(defensin)具有抵抗外來病原體的生物活性,在蜱類先天免疫系統(tǒng)中發(fā)揮重要作用。本研究以鈍刺血蜱為研究對象,通過基因克隆得到5條防御素基因序列,對其進(jìn)行生物信息學(xué)分析(蛋白序列、同源性、理化性質(zhì)以及三維結(jié)構(gòu)),并對原核表達(dá)的重組蛋白進(jìn)行功能研究(抑菌活性、抗氧化活性、溶血活性)。將5條基因分別命名為Hd-defensin-1、Hd-defensin-2、Hd-defensin-3、Hd-defensin-4、Hd-defensin-5,其開放閱讀框分別為246bp、225bp、225bp、210bp和225bp;編碼氨基酸個數(shù)為88、74、74、69和74;成熟肽長度為48、38、38、38和38;等電點大小為7.47、8.73、11.51、7.47和9.65。與NCBI中的不同蜱源防御素進(jìn)行比對,同源性均達(dá)到60%以上,其中Hd-defensin-1和Hd-defensin-2與長角血蜱的防御素(ABO28926.1)同源性最高,為95%。對Hd-defensin-3和Hd-defensin-5進(jìn)行原核表達(dá),獲得重組蛋白rHd-defensin-3和rHd-defensin-5,抑菌活性的研究發(fā)現(xiàn)rHd-defensin-3和rHd-defensin-5對革蘭氏陽性菌活性較強(qiáng),對部分陰性菌有活性,對真菌也存在一定的抗菌作用。熒光定量結(jié)果表明,在不同發(fā)育時期Hd-defensin-3在若蜱中的表達(dá)量最高,而各組織中在唾液腺的表達(dá)量最高,其次為中腸、卵巢、馬氏管;Hd-defensin-5在不同發(fā)育時期幼蜱和若蜱的表達(dá)量差異不顯著但均高于卵和成蜱期,各組織中在唾液腺表達(dá)量最高。菌脅迫檢測結(jié)果表明,Hd-defensin-3基因表達(dá)量與對照組比較差異不顯著;Hd-defensin-5表達(dá)量發(fā)生變化,且經(jīng)粘液菌脅迫后表達(dá)量最高。抗氧化能力和溶血活性檢測中,rHd-defensin-3和rHd-defensin-5對DPPH·和ABTS·+均有較強(qiáng)的清除能力并且對人類紅細(xì)胞未表現(xiàn)溶血作用。上述研究結(jié)果為新型抗菌和抗氧化藥劑的研發(fā)提供了重要參考。
[Abstract]:Haemaphysalis obliquus (Haemaphysalis doenitzi) is mainly distributed in Fujian, Yunnan and Taiwan in China. It is parasitic on the body surface of birds and small mammals, and can carry many pathogens such as Babes's, which causes serious harm to wildlife and human beings. Defensin (defensin) has biological activity against foreign pathogens and plays an important role in the innate immune system of ticks. In this study, five defensin gene sequences were obtained by gene cloning and bioinformatics analysis (protein sequence, homology). Physical and chemical properties and three-dimensional structure of the recombinant prokaryotic protein were studied (bacteriostatic activity, antioxidant activity, hemolytic activity). The five genes were named Hd-defensin-1,Hd-defensin-2,Hd-defensin-3,Hd-defensin-4,Hd-defensin-5,. The open reading frames were 246bpmm225bpn225bp and 225bp, respectively. The number of amino acids encoded was 8810bp and 74bp respectively. The length of mature peptide was 488rpm 38N38 and 38bp, and the isoelectric point was 7.478.733nd11.51c7.47 and 9.65bp. respectively. The homology of defensins from different ticks in NCBI was over 60%, and the homology between Hd-defensin-1 and Hd-defensin-2 was the highest (95%) with that of Haemaphysalis longicornis (ABO28926.1). After prokaryotic expression of Hd-defensin-3 and Hd-defensin-5, the antibacterial activity of recombinant protein rHd-defensin-3 and rHd-defensin-5, was obtained. It was found that rHd-defensin-3 and rHd-defensin-5 had strong activity against Gram-positive bacteria, partial negative bacteria and some fungi. The results of fluorescence quantitative analysis showed that the expression of Hd-defensin-3 was the highest in different developmental stages, and the highest in salivary gland, followed by midgut and ovary. The expression of Hd-defensin-5 in different developmental stages was not significant but higher than that in eggs and adult ticks, and the highest expression was found in salivary glands. The results of strain stress showed that the expression of Hd-defensin-3 gene was not significantly different from that of the control group, and the expression of Hd-defensin-5 was the highest under myxobacteria stress. Both rHd-defensin-3 and rHd-defensin-5 have strong scavenging ability to DPPH and ABTS in the determination of antioxidant capacity and hemolytic activity, but they have no hemolytic effect on human erythrocytes. These results provide an important reference for the research and development of new antimicrobial and antioxidant agents.
【學(xué)位授予單位】：河北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q966;Q78

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