钝刺血蜱防御素基因的克

发布时间：2018-10-09 18:24

【摘要】：钝刺血蜱(Haemaphysalis doenitzi)主要分布于中国的福建、云南、台湾等地,多寄生在鸟类和小型哺乳动物体表上,能携带巴贝斯虫等多种病原体,对野生动物和人类造成严重危害。防御素(defensin)具有抵抗外来病原体的生物活性,在蜱类先天免疫系统中发挥重要作用。本研究以钝刺血蜱为研究对象,通过基因克隆得到5条防御素基因序列,对其进行生物信息学分析(蛋白序列、同源性、理化性质以及三维结构),并对原核表达的重组蛋白进行功能研究(抑菌活性、抗氧化活性、溶血活性)。将5条基因分别命名为Hd-defensin-1、Hd-defensin-2、Hd-defensin-3、Hd-defensin-4、Hd-defensin-5,其开放阅读框分别为246bp、225bp、225bp、210bp和225bp;编码氨基酸个数为88、74、74、69和74;成熟肽长度为48、38、38、38和38;等电点大小为7.47、8.73、11.51、7.47和9.65。与NCBI中的不同蜱源防御素进行比对,同源性均达到60%以上,其中Hd-defensin-1和Hd-defensin-2与长角血蜱的防御素(ABO28926.1)同源性最高,为95%。对Hd-defensin-3和Hd-defensin-5进行原核表达,获得重组蛋白rHd-defensin-3和rHd-defensin-5,抑菌活性的研究发现rHd-defensin-3和rHd-defensin-5对革兰氏阳性菌活性较强,对部分阴性菌有活性,对真菌也存在一定的抗菌作用。荧光定量结果表明,在不同发育时期Hd-defensin-3在若蜱中的表达量最高,而各组织中在唾液腺的表达量最高,其次为中肠、卵巢、马氏管;Hd-defensin-5在不同发育时期幼蜱和若蜱的表达量差异不显著但均高于卵和成蜱期,各组织中在唾液腺表达量最高。菌胁迫检测结果表明,Hd-defensin-3基因表达量与对照组比较差异不显著;Hd-defensin-5表达量发生变化,且经粘液菌胁迫后表达量最高。抗氧化能力和溶血活性检测中,rHd-defensin-3和rHd-defensin-5对DPPH·和ABTS·+均有较强的清除能力并且对人类红细胞未表现溶血作用。上述研究结果为新型抗菌和抗氧化药剂的研发提供了重要参考。
[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.
【学位授予单位】：河北师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q966;Q78

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