多头带绦虫黏着蛋白家族基因克隆与功能鉴定

发布时间：2018-03-05 23:13

  本文选题：多头带绦虫　切入点：TmAdh　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：脑多头蚴病(Cerebral coenurosis)是由多头带绦虫(Taenia multiceps)的中绦期幼虫脑多头蚴(Coenurus cerebralis)引起的一种人畜共患寄生虫病。脑多头蚴可寄生于牛、羊等家畜的脑、脊髓等中枢神经系统,使宿主产生神经症状,甚至导致死亡,人作为中间宿主亦可偶发脑多头蚴病。因此,该病不仅给畜牧业造成巨大的经济损失,而且威胁着人体健康。随着分子生物学和免疫学技术的发展,使用疫苗防治绦虫蚴病取得了一定的成效,成为目前研究的热点。本研究以多头带绦虫基因组DNA为模板,PCR扩增Tm Adh基因家族成员基因组序列,获得6个含有N端信号肽和1个Fn III结构域的基因序列。利用生物信息学软件对Tm Adh家族成员进行预测分析,发现其都由3个外显子和2个内含子组成;序列比对显示其蛋白序列一致性在44.9-99.2%;进化分析发现该蛋白属于带科绦虫免疫保护性抗原家族的一个新的亚类。以脑多头蚴c DNA为模板扩增出Tm Adh1、Tm Adh2和Tm Adh4 c DNA基因片段,Tm Adh3基因片段人工合成,将Tm Adh1-4基因片段连接至原核表达载体p GEX4T-1进行原核表达。成功表达出产物大小约为39.0、38.1、38.9和37.8 ku的重组蛋白。利用Western-blot分析Tm Adh1-4重组蛋白的反应原性,结果表明脑多头蚴自然感染绵羊血清与Tm Adh3重组蛋白反应明显,而与Tm Adh1、Tm Adh2和Tm Adh4重组蛋白反应不明显。细胞黏着活性实验结果表明重组蛋白Tm Adh1-4对中国仓鼠卵巢细胞(CHO)和正常鼠肾细胞(NRK)都有黏着活性。免疫组化实验结果表明,Tm Adh1主要分布在脑多头蚴原头节的上皮、顶突(主要在小钩)、组织内部有零星分布;Tm Adh2主要分布在脑多头蚴原头节的组织内部、顶突、小钩、上皮有少量分布;Tm Adh3主要分布在脑多头蚴原头节的顶突、小钩、上皮、组织内部零星分布;Tm Adh4大量分布在脑多头蚴原头节的组织内部、顶突和小钩上。重组蛋白的动物免疫保护实验结果显示Tm Adh1、Tm Adh3和Tm Adh4重组蛋白免疫绵羊后抗体水平都显著上升,三免后达到峰值,攻虫后抗体水平开始逐渐下降。用多头带绦虫虫卵感染绵羊后,Tm Adh1重组蛋白免疫组绵羊的脑包囊数较对照组减少61.71%,Tm Adh3的减虫率为53.17%,Tm Adh4的减虫率为50.76%,从而表明Tm Adh1、Tm Adh3和Tm Adh4重组抗原可以诱导绵羊对多头带绦虫虫卵感染产生一定的抵抗力。本研究为进一步研制脑多头蚴病疫苗奠定基础。
[Abstract]:Cerebral coenurosis (Cerebral Coenensis) is a zoonotic parasitic disease caused by the middle stage larvae of Taenia multicepsm (Taenia multiceps), which can parasitize the central nervous system, such as the brain, spinal cord, and so on of cattle, sheep and other domestic animals. It causes neurotic symptoms and even death of the host. As an intermediate host, human may occasionally develop polycephalosis. Therefore, the disease not only causes great economic losses to animal husbandry, With the development of molecular biology and immunology, the use of vaccine to prevent and treat tapeworm echinococcosis has made some progress. In this study, genomic DNA of Taenia multiplex was used as template to amplify genomic sequence of TM Adh gene family. Six sequences containing N-terminal signal peptide and one Fn III domain were obtained. The members of TM Adh family were predicted and analyzed by bioinformatics software. It was found that all of them were composed of 3 exons and 2 introns. Sequence alignment showed that the protein sequence was consistent in 44.9-99.2. Evolutionary analysis showed that the protein belonged to a new subclass of the family of immune protective antigens of Taenia solium. TmAdh1Tm Adh2 and TM Adh4 c DNA were amplified by using c DNA as template. The gene fragment of TM Adh3 was synthesized by artificial synthesis. The TM Adh1-4 gene fragment was ligated to the prokaryotic expression vector p GEX4T-1 for prokaryotic expression. The recombinant proteins with the sizes of 39.0U 38.1U 38.9 and 37.8 ku were successfully expressed. The reactivity of TM Adh1-4 recombinant protein was analyzed by Western-blot. The results showed that the recombinant protein of TM Adh3 in sheep serum infected naturally with cerebral polycephalosis was significantly reactivity. The results of cell adhesion assay showed that the recombinant protein TM Adh1-4 had adhesion activity to both Chinese hamster ovary cells (Cho) and normal rat renal cells (NRK). TM Adh1 was mainly distributed in the epithelium of the procephalic ganglion of the cerebrum polycephalosis. In the parietal process (mainly in the small uncinate, in the tissue there are scattered distribution of TM Adh2 mainly in the tissue of the cerebrum metacercaria, the parietal process, the small uncinate, the epithelium have a little distribution of TM Adh3, mainly distributed in the parietal process, the small hook, the epithelium of the cerebrum polycephalosis, the parietal process, the small hook, the epithelium. Tm Adh4 was distributed sporadically in the tissues of cerebrum polycephalosis, parietal process and uncinate. The results of animal immunological protection test showed that the antibody levels of TmAdh1Tm Adh3 and TM Adh4 recombinant protein increased significantly after immunization with sheep, and the results showed that TmAdh1Tm Adh3 and TM Adh4 recombinant protein increased significantly after immunizing sheep with TmAdh1Tm Adh3 and TM Adh4 recombinant protein. After three exemptions, the peak value was reached. The antibody level began to decrease gradually after infection with Taenia multiceps eggs in sheep. The number of brain cyst of sheep immunized with Tm Adh1 recombinant protein was 61.71% lower than that of control group, and the worm reduction rate of TM Adh3 was 53.17%. The decrease rate of TM Adh4 was 50.76%, which indicated that TM Adh1TM was reduced. The recombinant antigens of Adh3 and TM Adh4 could induce the resistance of sheep to egg infection of Taenia multicephalus, which laid a foundation for the further development of the vaccine against cerebrum multicephalic echinococcosis.
【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】

中国期刊全文数据库 前1条

1 王安;双龙;李宏图;僧格仁钦;德格吉日乎;陶雪苹;董湘清;白明珠;伊茹;卓甘格图;;呼盟牧区羊多头蚴病综合防治措施研究[J];中国农业大学学报;1998年S2期

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本文编号：1572303