日本血吸虫：湖北钉螺人工传代及线粒体atp6基因多态性研究

发布时间：2018-03-11 14:04

本文选题：日本血吸虫　切入点：湖北钉螺　出处：《中南大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的 1.采用人工方法建立钉螺室内外生存的条件,获得日本血吸虫感染的湖北钉螺,以实现日本血吸虫的室内传代与保种。 2.研究湖南省日本血吸虫不同地域自然隔离群线粒体ATP合成酶FO亚单位6(atp6)基因部分序列的遗传多态性,为湖南不同地域自然隔离群日本血吸虫的遗传特性研究提供实验依据。方法 1.用尼龙绢筛收集日本血吸虫成熟虫卵,在适当的条件下进行毛蚴孵化。将钉螺与毛蚴按1：15～1：20的比例进行感染,以获得第一代人工感染性钉螺,再以第一代(F1代)人工感染性钉螺逸出的尾蚴感染实验动物,获取成熟虫卵并孵化毛蚴,然后继续感染钉螺,获得第二代(F2代)人工感染性钉螺、同样方法完成第三代(F3代)人工感染性钉螺、第四代(F4代)人工感染性钉螺的传代、…,在实验室内建立血吸虫的传代和保种技术与方法。同时模拟钉螺野外生存环境,建立斜坡型、秧田型和沟渠型三种自然生态养螺池,进行钉螺生存、养殖条件的观察、比较。 2.提取日本血吸虫基因组总DNA,以特异性引物对线粒体atp6基因进行PCR扩增,通过单链构象多态性技术(SSCP)筛选出差异带型并进行测序,用DNA Star 5.0及Mega 4.0软件进行比对分析。结果 1.成功模拟钉螺野外生存的环境,建立了斜坡型、秧田型、沟渠型三种类型的养螺池,分别投放钉螺3万、2万、4万只,三种类型的螺池中的钉螺均成功越冬并出现了新生幼螺,其中以斜坡型螺池生存条件最佳,其平均幼螺率达到17.17%,实现了人工模拟条件下钉螺的自身繁殖。室内人工传代研究共获得Fl代阳性钉螺796只,F2代阳性钉螺272只, F3代阳性钉螺362只,成功实现了日本血吸虫的室内传代与种质钉螺的保种。 2.湖南省5个流行区的日本血吸虫PCR扩增后获得了483 bpatp6部分序列,检测出17个变异位点,变异率为3.52%,雌雄虫之间的差异为0.0～1．3%,不同地理来源虫株间的差异率为0.0～1.5%。聚类分析结果表明：湖南省不同地域自然隔离群日本血吸虫分离株线粒体atp6基因的个体遗传差异明显,尤以泪罗和岳阳君山两地分离虫株表现突出。结论 1.人工模拟了野外养殖钉螺条件,建立了三种类型的养螺池,已可以满足日本血吸虫室内传代深入研究的需要。成功实现了日本血吸虫的F1代、F2代、F3代的室内传代,为血吸虫种群的遗传变异、溯源研究创造了条件。 2.湖南不同地域自然隔离群日本血吸虫线粒体atp6基因存在明显的个体差异,但是否由于生存环境的选择压力造成了同一地理来源日本血吸虫个体之间atp6基因的遗传差异,其原因有待于进一步研究。
[Abstract]:Purpose. 1. Artificial methods were used to establish the living conditions of Oncomelania hupensis in and out of the room, and to obtain Schistosoma japonicum infected Oncomelania hupensis in order to realize the indoor passage and species conservation of Schistosoma japonicum. 2. To study the genetic polymorphism of mitochondrial ATP synthase FO subunit (FO) in different regions of Schistosoma japonicum in Hunan Province, and to provide experimental basis for the study of genetic characteristics of Schistosoma japonicum in different regions of Hunan Province. Method. 1. The mature eggs of Schistosoma japonicum were collected by nylon silk screen and hatched under suitable conditions. The first generation of artificial infected snails were obtained by infecting the snails and the cercariae in the proportion of 1: 15 to 1: 20. The experimental animals were infected with cercariae escaped from artificial infected snails in the first generation (F1 generation). Mature eggs were obtained and hatched cercariae were hatched, and then continued infection of Oncomelania hupensis was carried out to obtain the second generation of artificial infected Oncomelania hupensis. Methods the third generation F 3 generation) artificial infected snail, 4th generation F 4 generation) artificial infected snail were subcultured... The breeding and conservation techniques and methods of Schistosoma japonicum were established in laboratory. Meanwhile, the field living environment of snail was simulated. Three kinds of natural ecological snail culture ponds were established, such as slope type, Yangtian type and ditch type. The survival and culture conditions of Oncomelania hupensis were observed and compared. 2. The total genomic DNA of Schistosoma japonicum was extracted, and the mitochondrial atp6 gene was amplified by PCR with specific primers. The differential bands were screened by single strand conformation polymorphism (SSCP) and sequenced. The results were compared with those of DNA Star 5.0 and Mega 4.0. Results. 1. Successfully simulating the living environment of snails in the field, establishing three types of snail culture ponds: slope type, rice field type and ditch type, and placing 30,000, 20,000 and 40,000 snails, respectively. The snails in the three types of snail ponds were successfully overwintering and the newborn snails appeared. Among them, the sloping snail pond has the best living conditions. The average juvenile snail rate was 17.17, and the self-propagation of Oncomelania hupensis was realized under artificial simulated conditions. 796 F1 generation positive snails and 362 F3 generation positive snails were obtained in laboratory. The indoor passage of Schistosoma japonicum and the conservation of germplasm snail were successfully realized. 2. PCR amplification of Schistosoma japonicum from 5 endemic areas of Hunan Province was carried out, and the partial sequence of 483 bpatp6 was obtained, and 17 mutation sites were detected. The variation rate was 3.52, the difference between female and male was 0.01.3 and the difference among different geographical sources was 0.01.5.The results of cluster analysis showed that the individual genetic difference of mitochondrial atp6 gene of Schistosoma japonicum isolates from different regions of Hunan Province was obvious. Especially in Yiluo and Yueyang Junshan isolated insect strains outstanding. Conclusion. 1. Artificial simulation of snail culture conditions in the field and establishment of three types of snail culture ponds can meet the need of further study on the indoor passage of Schistosoma japonicum, and successfully realize the indoor passage of F _ 1 and F _ 2 generation of Schistosoma japonicum. It provides conditions for genetic variation and traceability of Schistosoma japonicum population. 2. There are obvious individual differences in mitochondrial atp6 gene of Schistosoma japonicum in different regions of Hunan Province, but whether the genetic difference of atp6 gene among individuals from the same geographical origin is caused by the selection pressure of living environment. The reasons need to be further studied.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R184

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