传播疟疾的媒介按蚊遗传基因特性研究及抗菌肽的克

发布时间：2018-06-06 18:53

本文选题：随机扩增 + 按蚊　；参考：《安徽理工大学》2006年硕士论文

【摘要】：目的比较分析对约氏疟原虫易感的斯氏按蚊和不易感的大劣按蚊的基因组DNA的多态性，探讨媒介按蚊与疟原虫遗传基因间的相互关系以及为研究大劣按蚊的先天免疫，克隆大劣按蚊的抗菌肽defensins基因并对其进行序列分析。方法设计5条随机引物，应用随机扩增多态性DNA(Random amplified polymorphic DNA，RAPD)技术对斯氏按蚊、大劣按蚊的正常蚊、感染蚊及其幼虫的基因组DNA进行随机扩增，扩增产物经琼脂糖凝胶电泳后分析比较结果；并分别提取感染疟原虫和细菌的大劣按蚊的基因组DNA和总RNA，分别以特异引物和简并引物采用PCR和RT-PCR法扩增出目的基因片段，扩增的目的片段经纯化回收后克隆到PMD-18T中，转化入大肠杆菌DH 5α，经TA克隆后测序并进行序列分析。结果 5条随机引物对两种按蚊扩增的条带数不同，其中P5对斯氏按蚊扩增的效果较好，P4对大劣按蚊扩增效果较好，P1对大劣按蚊无扩增产物，用P3对两种按蚊扩增，感染前后的扩增产物电泳条带数相同，但其表达量不同；成蚊与幼虫带型数量不同；而疟原虫感染的大劣按蚊用简并引物经RT-PCR扩增出一条219bp目的片段，与埃及伊蚊defensins有95％同源性，基因组DNA用特异引物扩出一308bp目的片段，与冈比亚按蚊有100％同源性，细菌感染的大劣按蚊总RNA用特异引物扩增出一条333bp目的片段，与冈比亚按蚊有100％同源性，用简并引物扩出一条188bp目的片段，与冈比亚按蚊有87％同源性，与白蚊伊蚊defensinsD有88％同源性。结论：RAPD结果显示斯氏按蚊和大劣按蚊之间存在基因差异，同种未感染蚊和感染蚊基因带型相同，而表达量存在差异；幼虫与成虫的基因差异较大。成功克隆大劣按蚊抗菌肽defensins基因，其与冈比亚按蚊同源性较高。为进一步探讨按蚊对疟原虫感染的先天免疫反应，研究按蚊抗菌肽的生物活性奠定基础。
[Abstract]:Objective to compare and analyze the genetic polymorphisms of Anopheles stephensi and Anopheles dirus susceptible to Plasmodium yoelii, and to explore the relationship between the genetic genes of Anopheles vector and Plasmodium and to study the innate immunity of Anopheles dirus. The antimicrobial peptide defensins gene of Anopheles dirus was cloned and sequenced. Methods five random primers were designed and randomly amplified polymorphic DNA-random amplified polymorphic DNA (RAPDD) technique was used to detect Anopheles stephensi and Anopheles dirus. The genomic DNA of infected mosquitoes and their larvae were amplified randomly, and the results were analyzed and compared by agarose gel electrophoresis. The genomic DNA and total RNAs of Anopheles dirus infected with malaria parasites and bacteria were extracted, and the target gene fragments were amplified by PCR and RT-PCR with specific primers and degenerate primers, and the amplified fragments were cloned into PMD-18T after purification and recovery. DH5 伪 was transformed into Escherichia coli DH5 伪, sequenced and sequenced by TA cloning. Results the number of bands amplified by 5 random primers was different between two species of Anopheles. The amplification effect of P5 to Anopheles stephensi was better than that of P4 to Anopheles inferioris. There was no amplification product of P1 to Anopheles dirus. The number of electrophoresis bands of amplification products before and after infection was the same, but the expression of the products was different when using P3 to amplify the two species of Anopheles dirus. A 219bp fragment was amplified by RT-PCR from Anopheles malarial infected Anopheles dirus with 95% homology with Aedes aegypti defensins, and a 308bp fragment was amplified by specific primers. There was 100% homology with Anopheles gambiae. A specific primer was used to amplify a 333bp fragment from total Anopheles dirus infected by bacteria, and 100% homology with Anopheles gambiae. A 188bp fragment was amplified by degenerate primers. There was 87% homology with Anopheles gambiae and 88% homology with defensinsD of Aedes albopictus. However, there were significant differences in gene expression between larva and adult. The antimicrobial peptide defensins gene of Anopheles dirus was cloned successfully, which had high homology with Anopheles gambiae. In order to further study the innate immune response of Anopheles to Plasmodium infection and to study the biological activity of Anopheles repens antimicrobial peptide.
【学位授予单位】：安徽理工大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R384.1

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