本文選題：中華按蚊 + 差異表達　； 參考：《第二軍醫大學》2010年碩士論文

【摘要】： 蚊蟲感染瘧原蟲后可誘導蚊體內大量免疫相關基因的轉錄激活,進而干擾瘧原蟲在蚊體內的發育。因此許多研究者通過各種方法篩選其中的免疫相關基因,希望從中找到新的分子作為干擾蚊媒發育和傳病的靶點。而目前大部分工作還主要集中在非洲岡比亞按蚊。 中華按蚊分布廣,種群大,是我國傳播瘧疾的重要媒介。本研究旨在構建中華按蚊感染瘧原蟲后24小時的雙向cDNA消減文庫,從中篩選出抑制或促進蚊體內瘧原蟲發育的免疫相關基因及其他差異表達基因,與已有的岡比亞按蚊及其它蚊媒的基因組序列作同源性比對,并預測其功能,為后期的免疫基因功能驗證提供實驗資料。 采用抑制性消減雜交(SSH)技術構建了中華按蚊感染組(吸感染血后24小時)及對照組(吸正常血后24小時)的雙向cDNA消減文庫。最終從構建的正向文庫1(以感染組為測試組,對照組為驅動組)中獲得了15條單一序列,理論上指中華按蚊感染瘧原蟲以后較對照組高表達的基因；從構建的反向文庫2(以感染組為驅動組,對照組為測試組)中獲得了18條單一序列,理論上指中華按蚊感染瘧原蟲以后低表達的基因。將兩個庫中的序列與NCBI/tblastx/ESTs表達序列標簽數據庫、NCBI/blastx蛋白質數據庫,以及NCBI/blastn等作同源性搜索,結果顯示兩個文庫中共有19條序列可以找到相似度較高的疑似為同源性序列,還有14條序列未能夠比對到相似序列。 隨后,對所得序列進行了簡單的生物信息學預測分析。分別利用了信號肽預測軟件SignalPv3.0、跨膜螺旋結構預測軟件TMHMMv2.0和非經典分泌蛋白預測軟件SecretomeP及蛋白定位預測軟件targetp v1.1,初步結果顯示4條序列含有信號肽區域,6條序列含有跨膜螺旋結構,其中2條既有信號肽區域又具備跨膜螺旋結構；12條序列可能屬于非經典分泌蛋白；3條序列可能屬于線粒體靶位肽(mitochondrial targeting peptide,mTP),5條序列有可能屬于分泌通路信號肽(secretory pathway signal peptide,SP)。另外,使用ProtFun 2.2 predictions軟件對這些序列進行了功能上的注解,發現多數序列參與蛋白翻譯,能量代謝,轉錄調節,蛋白運輸及結合粘附等過程,Gene Ontology category分析結果顯示相關序列編碼的蛋白可能為生長因子,受體,載體,結構蛋白或者參與免疫反應的相關蛋白等。從這些結果可以看出,文庫里的基因不僅僅是與免疫相關,還有許多與免疫無關的差異表達基因。 本研究模擬相同條件重復了感染實驗,挑選文庫里部分基因通過real-time PCR方法進行了驗證,并采用RACE技術對驗證結果中感興趣的差異表達序列片段進行了全長擴增,獲得了中華按蚊熱休克蛋白40 (HSP40)和一條未知基因的cDNA全長序列,另外獲得了參與脂肪酸β-氧化的關鍵酶-中華按蚊烯酰輔酶A水合酶(enoyl CoA hydratase, ECAH)的896 bp的3′端序列及另外2條未知基因的3′端序列,長度分別為911 bp和662 bp。 本實驗作為研究中華按蚊感染與免疫的分子機制是國內的首次初步嘗試,獲得的結果尚需進一步功能驗證。系統地研究中華按蚊免疫相關和代謝相關,甚至于其他功能相關的分子還需要較長時間的探索。本次實驗結果希望為后期進行的驗證基因(特別是未知基因)功能方面的實驗奠定良好的基礎,在對其充分的研究后,從中找到可被運用的分子靶點,為控制傳瘧蚊媒的新策略提供實驗材料。
[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.
【學位授予單位】：第二軍醫大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R384.1

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