残缘璃眼蜱subolesin和半胱氨酸蛋白酶基因生物学功能初步研究
发布时间：2018-07-28 18:02
【摘要】：残缘璃眼蜱作为专性吸血外寄生虫,能够传播多种病原体(特别是环形泰勒虫),对畜牧业造成巨大的经济损失。目前对于蜱以及蜱传病的防治方法主要以化学药物治疗为主,但由此引发的环境污染、蜱的抗药性等问题越来越严重,因此免疫学防治方法逐渐被重视起来。免疫学方法重要的基础是寻找合适的蜱的功能基因作为保护性抗原,为抗蜱及蜱传病疫苗提供理论性基础。根据已有的蜱种功能基因的研究表明,subolesin和半胱氨酸蛋白酶基因存在于多种生物体中;subolesin基因在高等真核生物中具有较高的同源性,在蜱种中具有较好的保守性;半胱氨酸蛋白酶(CP)与宿主血液消化及蜱的生长发育相关。试验首次从残缘璃眼蜱、亚洲璃眼蜱中扩增出subolesin基因ORF,大小均为492 bp,编码163个氨基酸。生物信息学分析表明,subolesin基因在各蜱种间具有高度保守性,且有一定的种间特异性和属间差异性。将subolesin基因连接到pGEX-4T-1载体上进行重组表达,重组蛋白大小约为45 ku。通过SDS-PAGE和Western blotting分析显示,体外表达重组蛋白以包涵体形式存在,纯化的目的蛋白与兔抗残缘璃眼蜱饥饿成蜱血清、抗蜱唾液腺和抗蜱卵巢血清发生免疫反应。实时荧光定量分析表明,subolesin基因存在于蜱的各个发育阶段和组织,但相对表达丰度不同,中肠、唾液腺的表达丰度相对较高,在蜱吸血前后表达丰度有很大差异,成蜱吸血过程中subolesin基因表达丰度出现先上调后下调的现象;动物保护试验和RNAi试验证明,蜱的吸血率、饱血体重、饱血时间、产卵率、卵的孵化率都受到影响;这些现象表明该基因可能与蜱的生长、发育及吸血相关。本试验利用通用引物cys500首次从残缘璃眼蜱中获得CP保守序列,通过5'RACE和3'RACE扩增技术获得CP基因全长。CP基因全长为1192 bp,包含一个999 bp的ORF。生物学软件分析CP基因在不同蜱种间具有一定的保守性。连接到pET30a载体上成功表达,重组蛋白约为45 ku;SDS-PAGE和Western blotting分析显示,体外表达的融合蛋白以包涵体的形式存在,并和兔抗残缘璃眼蜱饥饿成蜱血清发生免疫反应,表明该融合蛋白具有良好的反应原性。实时荧光定量分析显示,CP基因表达具有选择性,组织中仅中肠有较高的表达丰度,其他组织中相对表达丰度较低,基本可以忽略,蜱卵中该基因相对表达丰度较高,蜱吸血过程中基因表达丰度出现上调现象;动物保护性试验和RNAi结果表明:蜱的吸血、饱血程度及产卵情况受到影响,证明CP可能与蜱的生长、发育及卵发育相关。
[Abstract]:As a specific blood sucking parasite, ticks can transmit a variety of pathogens (especially Taylor ringworm), causing great economic losses to animal husbandry. At present, the main control methods for ticks and tick-borne diseases are chemotherapeutic treatment. However, the environmental pollution and the resistance of ticks are becoming more and more serious, so immunological methods have been paid more and more attention. The important basis of immunological methods is to find suitable functional genes of ticks as protective antigens and to provide theoretical basis for anti-tick and tick-borne vaccines. According to the existing functional genes of ticks, subolesin and cysteine protease genes were found to have high homology in higher eukaryotes and good conserved in ticks. Cysteine protease (CP) is associated with host blood digestion and the growth and development of ticks. For the first time, the subolesin gene ORF was amplified from ticks residualis and ticks asiatica, with a size of 492bp, encoding 163 amino acids. Bioinformatics analysis showed that the subolesin gene was highly conserved among the ticks, and had some interspecific and intergeneric differences. The subolesin gene was ligated into the pGEX-4T-1 vector for recombinant expression, and the size of the recombinant protein was about 45 ku. SDS-PAGE and Western blotting analysis showed that the recombinant protein expressed in vitro existed in the form of inclusion body. The purified target protein reacted with the sera of adult ticks, anti-tick salivary glands and anti-tick ovaries in rabbits. Real-time fluorescence quantitative analysis showed that the subolesin gene existed in various stages and tissues of ticks, but the relative expression abundance was different, and the expression abundance of midgut and salivary gland was relatively high. The expression abundance of subolesin gene was up-regulated and then down-regulated in adult ticks, the animal protection test and RNAi test showed that the rate of blood absorption, body weight, time, oviposition rate and hatching rate of ticks were all affected. These phenomena suggest that the gene may be related to the growth, development and blood absorption of ticks. The CP conserved sequence was obtained by using universal primer cys500 for the first time. The total length of CP gene was 1192 BP, including a 999bp ORF by 5'RACE and 3'RACE amplification. The analysis of CP gene by biological software was conserved among different ticks. The fusion protein was expressed in the form of inclusion body and immunoreactive with adult ticks sera from rabbits. The recombinant protein was about 45ku-SDS-PAGE and Western blotting analysis showed that the fusion protein expressed in vitro existed in the form of inclusion body. The results showed that the fusion protein had good reactivity. The results of real-time fluorescence quantitative analysis showed that the expression of CP gene was selective, only in the midgut, but in other tissues was relatively low, which could be neglected, and the relative expression abundance of the gene in ticks eggs was higher than that in other tissues. The results of animal protective test and RNAi showed that the degree of blood intake and oviposition of ticks were affected, which suggested that CP might be related to the growth, development and egg development of ticks.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
[Abstract]:As a specific blood sucking parasite, ticks can transmit a variety of pathogens (especially Taylor ringworm), causing great economic losses to animal husbandry. At present, the main control methods for ticks and tick-borne diseases are chemotherapeutic treatment. However, the environmental pollution and the resistance of ticks are becoming more and more serious, so immunological methods have been paid more and more attention. The important basis of immunological methods is to find suitable functional genes of ticks as protective antigens and to provide theoretical basis for anti-tick and tick-borne vaccines. According to the existing functional genes of ticks, subolesin and cysteine protease genes were found to have high homology in higher eukaryotes and good conserved in ticks. Cysteine protease (CP) is associated with host blood digestion and the growth and development of ticks. For the first time, the subolesin gene ORF was amplified from ticks residualis and ticks asiatica, with a size of 492bp, encoding 163 amino acids. Bioinformatics analysis showed that the subolesin gene was highly conserved among the ticks, and had some interspecific and intergeneric differences. The subolesin gene was ligated into the pGEX-4T-1 vector for recombinant expression, and the size of the recombinant protein was about 45 ku. SDS-PAGE and Western blotting analysis showed that the recombinant protein expressed in vitro existed in the form of inclusion body. The purified target protein reacted with the sera of adult ticks, anti-tick salivary glands and anti-tick ovaries in rabbits. Real-time fluorescence quantitative analysis showed that the subolesin gene existed in various stages and tissues of ticks, but the relative expression abundance was different, and the expression abundance of midgut and salivary gland was relatively high. The expression abundance of subolesin gene was up-regulated and then down-regulated in adult ticks, the animal protection test and RNAi test showed that the rate of blood absorption, body weight, time, oviposition rate and hatching rate of ticks were all affected. These phenomena suggest that the gene may be related to the growth, development and blood absorption of ticks. The CP conserved sequence was obtained by using universal primer cys500 for the first time. The total length of CP gene was 1192 BP, including a 999bp ORF by 5'RACE and 3'RACE amplification. The analysis of CP gene by biological software was conserved among different ticks. The fusion protein was expressed in the form of inclusion body and immunoreactive with adult ticks sera from rabbits. The recombinant protein was about 45ku-SDS-PAGE and Western blotting analysis showed that the fusion protein expressed in vitro existed in the form of inclusion body. The results showed that the fusion protein had good reactivity. The results of real-time fluorescence quantitative analysis showed that the expression of CP gene was selective, only in the midgut, but in other tissues was relatively low, which could be neglected, and the relative expression abundance of the gene in ticks eggs was higher than that in other tissues. The results of animal protective test and RNAi showed that the degree of blood intake and oviposition of ticks were affected, which suggested that CP might be related to the growth, development and egg development of ticks.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
【参考文献】
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1 吕文顺,张平成,魏宝瑛,黄守云,俞生荣,蒲正w，
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