本文選題：日本血吸蟲(chóng) + 四次跨膜蛋白　； 參考：《北京協(xié)和醫(yī)學(xué)院》2010年博士論文

【摘要】：血吸蟲(chóng)病嚴(yán)重威脅人類的健康,全球76個(gè)發(fā)展中國(guó)家仍有血吸蟲(chóng)病的流行,約有2億血吸蟲(chóng)病患者。血吸蟲(chóng)體被(tegument)是蟲(chóng)體與宿主直接接觸的表面,介導(dǎo)與宿主相互作用。對(duì)血吸蟲(chóng)表膜(tegument surface membrane)蛋白的研究,有助于了解血吸蟲(chóng)免疫逃避、免疫調(diào)節(jié)、營(yíng)養(yǎng)攝取等機(jī)制,進(jìn)而有可能發(fā)現(xiàn)新的干涉靶點(diǎn)。血吸蟲(chóng)體被存在著表達(dá)廣泛且多樣的四次跨膜蛋白家族(tetraspanin,TSP),曼氏血吸蟲(chóng)四次跨膜蛋白家族成員被認(rèn)為是具有保護(hù)潛力的抗原分子,其中Sm-TSP-2更是獲得高達(dá)～60%的保護(hù)力；而其在日本血吸蟲(chóng)中的同源分子Sj-TSP-2且存在廣泛的多態(tài)性,與免疫逃避相關(guān),提示這兩個(gè)物種在進(jìn)化過(guò)程中發(fā)生的歧化作用。因此,有必要對(duì)日本血吸蟲(chóng)的四次跨膜蛋白家族成員展開(kāi)深入研究,希望找到針對(duì)日本血吸蟲(chóng)的新的抗原和藥物靶點(diǎn)。 本研究首先利用生物信息學(xué)方法在日本血吸蟲(chóng)中共鑒定了29個(gè)四次跨膜蛋白家族(Sj-TSP)成員。其中,除Sj-tsp-2和Sj-25外,新發(fā)現(xiàn)3個(gè)Sj-tsp基因存在不同程度的變異體；另外,2個(gè)Sj-tsp存在選擇性剪接,提示可能參與血吸蟲(chóng)逃避宿主免疫反應(yīng)的過(guò)程,不適合作為抗原候選分子。 實(shí)時(shí)熒光定量PCR表明不同Sj-tsp基因在尾蚴、肺期童蟲(chóng)、雌雄成蟲(chóng)和蟲(chóng)卵中呈多態(tài)性轉(zhuǎn)錄；肺期童蟲(chóng)是對(duì)宿主免疫攻擊最敏感的發(fā)育期,這一時(shí)期Sj-tsp基因的表達(dá)受到關(guān)注。其中,12種Sj-tsp基因在童蟲(chóng)的相對(duì)較高水平表達(dá),(相對(duì)tubulin1),7種Sj-tsp基因的表達(dá)在尾蚴入侵宿主后呈顯著增加,說(shuō)明這7種分子對(duì)以適應(yīng)宿主體內(nèi)環(huán)境發(fā)揮一定的作用,但其中2種是變異基因分別是Sj-tsp-2和Sj25,另有1種為Sj-tsp-7存在選擇性剪接,被認(rèn)為是Sj-tsp基因既要在宿主體內(nèi)發(fā)揮功能,又要逃避宿主攻擊所采取的策略。 體外對(duì)保守且童蟲(chóng)期表達(dá)的19個(gè)Sj-tsp基因成功進(jìn)行RNAi,并探討其對(duì)蟲(chóng)體的潛在影響。其中,4種Sj-tsp基因的沉默效率在80%以上,9種Sj-tsp基因的沉默效率在40%-60%之間。但以上Sj-tsp抑制以后,童蟲(chóng)的表膜完整性不受影響,且生存率與對(duì)照組相比無(wú)顯著差異,提示Sj-tsp基因可能在體內(nèi)發(fā)揮與宿主相互作用的功能,而體外則檢測(cè)不到其抑制后對(duì)童蟲(chóng)的影響。綜合以上結(jié)果,基于Sj-tsp家族成員的保守性,分期轉(zhuǎn)錄水平、大親水環(huán)半胱氨酸數(shù)目等分子特征,我們初步篩選出5個(gè)具有潛力的Sj-tsp基因,為進(jìn)一步的免疫保護(hù)試驗(yàn)奠定基礎(chǔ)。 瘧疾是當(dāng)今世界對(duì)人類危害最為嚴(yán)重的傳染性疾病之一。全球每年有3億-5億人感染惡性瘧,有100萬(wàn)-300萬(wàn)人因這一疾病而死亡,其中大多數(shù)為非洲撒哈拉以南地區(qū)5歲以下的兒童。近年來(lái),由于耐藥瘧原蟲(chóng)株和耐藥蚊媒的不斷產(chǎn)生和擴(kuò)散,使瘧疾控制形勢(shì)更為嚴(yán)峻。因此,研制安全有效的瘧疾疫苗,成為控制瘧疾的重要手段。 本課題組前期工作借鑒了分子育種技術(shù)(即DNA改組)的隨機(jī)重組原理,利用表位改組技術(shù)構(gòu)建了多表位基因庫(kù),并用庫(kù)免疫血清篩選出高抗原性的陽(yáng)性克隆,發(fā)現(xiàn)其中VR312(改名為M.RCAg-1)基因克隆不僅能在小鼠模型誘導(dǎo)交叉免疫保護(hù)而且能在家兔模型中誘導(dǎo)抗惡性瘧原蟲(chóng)抑制性抗體；D10基因克隆在家兔模型中誘導(dǎo)抗惡性瘧原蟲(chóng)抑制性抗體。 本實(shí)驗(yàn)通過(guò)采用優(yōu)化的DNA真核表達(dá)載體、電穿孔的免疫途徑,結(jié)合4-1 BBL和西咪替丁這兩種佐劑等方面改進(jìn)DNA疫苗的免疫效果。結(jié)果發(fā)現(xiàn)電穿孔的免疫途徑在低劑量時(shí)即可誘導(dǎo)高水平的抗體滴度；pVAX1表達(dá)載體的免疫效果比VR1012表達(dá)載體較好,但4-1BBL和西咪替丁這兩種佐劑均不能有效提高M(jìn).RCAg-1和D10 DNA疫苗單獨(dú)免疫在小鼠體內(nèi)誘導(dǎo)的抗體滴度。
[Abstract]:Schistosomiasis is a serious threat to human health, and there is still a epidemic of schistosomiasis in 76 developing countries around the world. There are about 200 million schistosomiasis patients. Schistosoma (tegument) is a surface that is a direct contact between the host and host, and mediates the interaction with the host. The study of the tegument surface membrane protein can help to understand the blood sucking. The mechanism of insect immune escape, immunomodulation, nutrition intake and so on, and then the possible discovery of new interference targets. Schistosoma bodies are present in a wide and diverse expression of the four transmembrane protein family (tetraspanin, TSP). Members of the four transmembrane protein family of Schistosoma mansoni are considered as protective potential antigen molecules, of which Sm-TSP-2 is obtained. The protection force of up to 60% is up to 60%, and its homologous molecule in Schistosoma japonicum has a wide variety of polymorphism, which is related to the immune escape, suggesting the disproportionation of the two species in the evolution process. Therefore, it is necessary to further study the four transmembrane protein family members of Schistosoma japonicum, hoping to find the target for Japan. The new antigen and drug target of Schistosoma.
In this study, 29 four transmembrane protein family (Sj-TSP) members of Schistosoma japonicum were identified by bioinformatics. In addition to Sj-tsp-2 and Sj-25, 3 Sj-tsp genes were found to have varying degrees of variant. In addition, 2 Sj-tsp had selective splicing, suggesting that it might be involved in Schistosoma to escape the host immune response. The process is not suitable as an antigen candidate.
Real time fluorescence quantitative PCR showed that different Sj-tsp genes were polymorphic in the cercariae, the lung stage children, the male and female adults and the eggs, and the lung stage was the most sensitive development period for the host immune attack, and the expression of the Sj-tsp gene was concerned at this time. Among them, 12 Sj-tsp genes were expressed at relatively high levels of the child, (relative tubulin1), and 7 species. The expression of Sj-tsp gene is significantly increased after the invasion of the cercariae, indicating that these 7 molecules play a role in adapting to the host environment, but 2 of them are Sj-tsp-2 and Sj25, respectively, and the other 1 are selective splicing for Sj-tsp-7. It is considered that the Sj-tsp gene is not only functioning in the host, but also escaping from the host. A strategy taken by a host attack.
In vitro, 19 Sj-tsp genes expressed in the conservative and child stage were successfully carried out with RNAi, and the potential effect on the insect body was explored. Among them, the silencing efficiency of the 4 Sj-tsp genes was more than 80% and the silence efficiency of the 9 Sj-tsp genes was between 40%-60%. There is no significant difference, suggesting that the Sj-tsp gene may play a function with the host in vivo, but in vitro it can not detect the effect of its inhibition on the child. Based on the conservatism of the members of the Sj-tsp family, the level of transcriptional transcription, the number of large hydrocysteine, we have preliminarily screened 5 of them. The Sj-tsp gene of the force lays the foundation for further immune protection test.
Malaria is one of the most serious infectious diseases in the world today. 300 million -5 million people worldwide are infected with falciparum malaria every year, and 1 million -300 million people die of this disease. Most of them are children under the age of 5 in sub Saharan Africa. In recent years, the emergence and proliferation of drug resistant strains of malaria parasites and resistant mosquito vectors have been developed in recent years. The malaria control situation is even more serious. Therefore, developing a safe and effective malaria vaccine has become an important means to control malaria.
In the previous work, we used the principle of random recombination of molecular breeding technology (DNA regrouping), and constructed a multi epitope gene pool using epitope modification technique, and screened the positive clones with high antigenicity with the immune sera of the library. It was found that VR312 (renamed M.RCAg-1) gene clones could not only induce cross immunization protection in mice model but also in mice model. It can induce anti Plasmodium falciparum antibody in rabbit model, and clone D10 gene in rabbit model to induce anti Plasmodium falciparum inhibitory antibody.
In this experiment, the immune effects of the DNA vaccine were improved by using the optimized DNA eukaryotic expression vector, the immune pathway of electroporation, combined with the two adjuvant such as 4-1 BBL and cimetidine. The results showed that the immune pathway of electroporation could induce the high level of antibody titer at low dose, and the immune effect of the pVAX1 expression vector was more than the VR1012 expression. The vectors were good, but the two adjuvants of 4-1BBL and cimetidine could not effectively enhance the antibody titer induced by M.RCAg-1 and D10 DNA vaccine alone.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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