中华按蚊感染约氏疟原虫后24小时差异表达基因的筛选及分析

发布时间：2018-06-14 22:38

本文选题：中华按蚊 + 差异表达　；参考：《第二军医大学》2010年硕士论文

【摘要】： 蚊虫感染疟原虫后可诱导蚊体内大量免疫相关基因的转录激活,进而干扰疟原虫在蚊体内的发育。因此许多研究者通过各种方法筛选其中的免疫相关基因,希望从中找到新的分子作为干扰蚊媒发育和传病的靶点。而目前大部分工作还主要集中在非洲冈比亚按蚊。中华按蚊分布广,种群大,是我国传播疟疾的重要媒介。本研究旨在构建中华按蚊感染疟原虫后24小时的双向cDNA消减文库,从中筛选出抑制或促进蚊体内疟原虫发育的免疫相关基因及其他差异表达基因,与已有的冈比亚按蚊及其它蚊媒的基因组序列作同源性比对,并预测其功能,为后期的免疫基因功能验证提供实验资料。采用抑制性消减杂交(SSH)技术构建了中华按蚊感染组(吸感染血后24小时)及对照组(吸正常血后24小时)的双向cDNA消减文库。最终从构建的正向文库1(以感染组为测试组,对照组为驱动组)中获得了15条单一序列,理论上指中华按蚊感染疟原虫以后较对照组高表达的基因；从构建的反向文库2(以感染组为驱动组,对照组为测试组)中获得了18条单一序列,理论上指中华按蚊感染疟原虫以后低表达的基因。将两个库中的序列与NCBI/tblastx/ESTs表达序列标签数据库、NCBI/blastx蛋白质数据库,以及NCBI/blastn等作同源性搜索,结果显示两个文库中共有19条序列可以找到相似度较高的疑似为同源性序列,还有14条序列未能够比对到相似序列。随后,对所得序列进行了简单的生物信息学预测分析。分别利用了信号肽预测软件SignalPv3.0、跨膜螺旋结构预测软件TMHMMv2.0和非经典分泌蛋白预测软件SecretomeP及蛋白定位预测软件targetp v1.1,初步结果显示4条序列含有信号肽区域,6条序列含有跨膜螺旋结构,其中2条既有信号肽区域又具备跨膜螺旋结构；12条序列可能属于非经典分泌蛋白；3条序列可能属于线粒体靶位肽(mitochondrial targeting peptide,mTP),5条序列有可能属于分泌通路信号肽(secretory pathway signal peptide,SP)。另外,使用ProtFun 2.2 predictions软件对这些序列进行了功能上的注解,发现多数序列参与蛋白翻译,能量代谢,转录调节,蛋白运输及结合粘附等过程,Gene Ontology category分析结果显示相关序列编码的蛋白可能为生长因子,受体,载体,结构蛋白或者参与免疫反应的相关蛋白等。从这些结果可以看出,文库里的基因不仅仅是与免疫相关,还有许多与免疫无关的差异表达基因。本研究模拟相同条件重复了感染实验,挑选文库里部分基因通过real-time PCR方法进行了验证,并采用RACE技术对验证结果中感兴趣的差异表达序列片段进行了全长扩增,获得了中华按蚊热休克蛋白40 (HSP40)和一条未知基因的cDNA全长序列,另外获得了参与脂肪酸β-氧化的关键酶-中华按蚊烯酰辅酶A水合酶(enoyl CoA hydratase, ECAH)的896 bp的3′端序列及另外2条未知基因的3′端序列,长度分别为911 bp和662 bp。本实验作为研究中华按蚊感染与免疫的分子机制是国内的首次初步尝试,获得的结果尚需进一步功能验证。系统地研究中华按蚊免疫相关和代谢相关,甚至于其他功能相关的分子还需要较长时间的探索。本次实验结果希望为后期进行的验证基因(特别是未知基因)功能方面的实验奠定良好的基础,在对其充分的研究后,从中找到可被运用的分子靶点,为控制传疟蚊媒的新策略提供实验材料。
[Abstract]:The infection of malaria parasites in mosquitoes can induce the activation of a large number of immune related genes in the mosquitoes and interfere with the development of malaria parasites in the mosquitoes. Therefore, many researchers have screened the immune related genes through various methods, hoping to find new molecules as a target for interfering with mosquito breeding and disease transmission. It is mainly concentrated in Anopheles Anopheles Gambia.
Anopheles sinensis is an important medium for spreading malaria in China. This study aims to construct a bi-directional cDNA subtractive library of Anopheles sinensis infected by Anopheles sinensis for 24 hours after infection. The immune related genes and other differentially expressed genes are screened from the Anopheles sinensis to inhibit or promote the development of malaria parasites in the mosquitoes, and the existing Anopheles Gambia and other mosquito vectors The sequence of the genome was homologous and predicted its function, providing experimental data for functional verification of immune genes in later stage.
A bi-directional cDNA subtractive library of Anopheles sinensis infection group (24 hours after infection of the infected blood) and the control group (24 hours after normal blood) was constructed by the inhibitory subtractive hybridization (SSH) technique. Finally, 15 single sequences were obtained from the constructed positive library 1 (the infection group as the test group and the control group). In theory, the Anopheles sinensis was infected with malaria. 18 single sequences were obtained from the constructed reverse library 2 (the infection group as the driving group and the control group), which theoretically refers to the low expression of the Anopheles sinensis after the infection of the malaria parasite. The sequence of the two libraries and the NCBI/ tblastx/ESTs expression sequence label database, the NCBI/blastx egg The white matter database, as well as NCBI/blastn and other homologous searches, show that there are 19 sequences in two libraries that can find similar homologous sequences with higher similarity, and 14 sequences are not comparable to similar sequences.
Then, a simple bioinformatics prediction analysis was carried out. The signal peptide prediction software SignalPv3.0, the transmembrane spiral structure prediction software TMHMMv2.0, the non classic secretory protein prediction software SecretomeP and the protein positioning prediction software targetp v1.1 were used respectively. The preliminary results showed that the 4 sequences contained the signal peptide region and the 6 sequence. The column contains a transmembrane spiral structure, 2 of which have both signal peptide region and transmembrane spiral structure; the 12 sequence may belong to the non classical secretory protein; the 3 sequence may belong to the mitochondrial target peptide (mitochondrial targeting peptide, mTP), and the 5 sequence may belong to the secretory signaling peptide (secretory pathway signal peptide, SP). In addition, these sequences were functional annotated with ProtFun 2.2 predictions software, and most sequences were found to be involved in the process of protein translation, energy metabolism, transcription regulation, protein transport and binding adhesion. The Gene Ontology category analysis showed that the related sequence encoded proteins may be growth factors, receptors, carriers and structural eggs. White or involved in the related proteins of the immune response. From these results, we can see that the genes in the library are not only related to the immune system, but also a number of differentially expressed genes that are not related to the immune system.
In this study, the infection experiment was repeated on the same condition, and some genes in the selected library were tested by real-time PCR method, and the RACE technique was used to amplify the full length of the differential expression sequences in the verifying results, and the cDNA full length sequence of the heat shock protein 40 (HSP40) and an unknown gene of Anopheles sinensis was obtained. In addition, the 896 BP 3 'terminal sequence of the Anopheles sinensis coenzyme A hydrate enzyme (enoyl CoA hydratase, ECAH) and the 3' terminal sequence of the other 2 unknown genes were obtained, and the length was 911 BP and 662 bp., respectively.
This experiment is the first preliminary attempt to study the molecular mechanism of Anopheles sinensis infection and immunity in China. The results need further functional verification. It is necessary to systematically study the immune related and metabolic related factors of Anopheles sinensis and even other functional related molecules for a long time. The results of this experiment hope to be carried out later. It lays a good foundation for the experiment of the function of the verifying genes (especially the unknown genes). After the full study, we find the molecular targets that can be used and provide experimental materials for the new strategies for the control of malaria vector.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R384.1

【参考文献】