本文關(guān)鍵詞： 斑馬魚 腸道凝集素 模式識別 糖識別結(jié)構(gòu)域 先天免疫　出處：《山東大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：免疫系統(tǒng)最根本的作用是識別“自己”和“非己”,先天性免疫是免疫的第一道防線,先天性免疫系統(tǒng)對于“自己”和“非己”的識別主要是由一系列基因編碼受體來完成的,這些受體被稱為模式識別受體(PRRs)。病原分子共有的成分,比如細胞壁的多糖以及RNA病毒的雙鏈等,能夠被PRRs識別,并引發(fā)一系列的信號通路來對各種免疫相關(guān)基因的表達進行調(diào)控,從而達到清除病原體的效果。凝集素(Lectin)不僅能夠直接參與免疫防御,更是一種重要的模式識別分子,在先天性免疫應(yīng)答中發(fā)揮重要作用。腸道凝集素(Intelectin, ITLN)是近年來新發(fā)現(xiàn)的一種能夠結(jié)合β-呋喃半乳糖的凝集素,在多種生命活動中發(fā)揮重要作用,并且與人類的很多疾病如哮喘、心臟病、腸炎、慢性阻塞性肺氣腫甚至癌癥都有一定關(guān)系。目前關(guān)于腸道凝集素的研究主要集中在哺乳動物、兩棲動物、硬骨魚、文昌魚和海鞘當中。雖然不同物種的ITLN序列高度保守,但是它們在表達模式和功能上也存在一定差異。鑒于ITLN在模式識別中的重要功能,研究其模式識別的機制顯得尤為重要。斑馬魚作為一種重要的模式生物,具有較為完整的先天免疫和后天免疫系統(tǒng),同時其免疫系統(tǒng)在分子和細胞水平上都與哺乳動物相似度很高。所以我們選擇了斑馬魚的腸道凝集素(zebrafish Intelectin, zITLN)來研究其模式識別的機制。我們首先利用生物信息學(xué)的技術(shù)手段,對斑馬魚Intelectin的進化地位進行分析,選擇了處于三個不同分支上的斑馬魚Intelectin:zITLN1、zITLN2和zITLN3來進行研究。我們對這3個zITLN進行了克隆,利用實時熒光定量PCR(Real-time PCR)的方法對成體斑馬魚的zITLN1、zITLN2和zITLN3的組織分布進行了探索,實驗結(jié)果表明3個zITLN在腸道、眼球、皮膚、鰓、肝臟以及尾鰭中均有表達,但表達模式及表達量不盡相同：zITLN1和zITLN2主要集中在腸道中表達,肝臟中表達量很低,而zITLN3與前兩者不同,主要集中在肝臟中表達,當然其在腸道中的表達量也相對較高。我們同樣利用Real-time PCR的方法檢測了金黃色葡萄球菌對斑馬魚Intelectin表達的調(diào)控,結(jié)果表明金黃色葡萄球菌可以引起zITLN1、zITLN2和zITLN3的表達上調(diào),但是程度與模式不盡相同：zITLN1和zITLN3對于金黃色葡萄球菌的刺激反應(yīng)更為靈敏,在注射金黃色葡萄球菌之后的4小時就出現(xiàn)了表達的上調(diào),8小時達到最高值,而zITLN2在前8小時中的上調(diào)很緩慢,直到10小時突然達到峰值。我們利用大腸桿菌表達系統(tǒng)對3種ITLN進行了表達,并對其表達產(chǎn)物進行純化。細菌凝集實驗中,熒光鏡檢顯示,鈣離子存在時,zITLN1、zITLN2和ZITLN3重組蛋白均可以不同程度的引起FITC標記的細菌的凝集,zITLN1和zITLN3一樣凝集金黃色葡萄球菌的能力要強于凝集大腸桿菌的能力,而zITLN2對于兩種細菌的凝集卻沒有表現(xiàn)出明顯差異。Western blotting結(jié)果顯示3個ITLN都可以結(jié)合細菌,zITLN1和zITLN3一樣結(jié)合金黃色葡萄球菌的能力要強于結(jié)合大腸桿菌的能力,但是ZITLN2對于兩種細菌的結(jié)合沒有表現(xiàn)出明顯差異。ELISA定量分析的結(jié)果表明zITLN1和zITLN3對PGN的親和性要高于LPS,而zITLN2在多糖結(jié)合上沒有明顯的偏向性�？偲饋碚f,zITLN2結(jié)合多糖的能力要高于zITLN1和zITLN3。上述結(jié)果表明：zITLN1、zITLN2和zITLN3可以識別細菌細胞壁的主要成分,從而能結(jié)合細菌并引發(fā)細菌的凝集。但是它們的識別和結(jié)合效應(yīng)以及后續(xù)的作用機制可能不同。為了進一步探究intelectin基因內(nèi)在的糖識別結(jié)構(gòu)域,我們構(gòu)建了zITLN1-D5、zITLN2-D5和ZITLN3-D5三個缺失突變體,通過細菌凝集、細菌結(jié)合和多糖結(jié)合的實驗在斑馬魚當中首次發(fā)現(xiàn)zITLN序列C末端100個左右的氨基酸(D5)在細菌凝集、細菌結(jié)合和糖結(jié)合方面的功能與其全長并無明顯差異。說明ITLN中負責多糖識別的是D5結(jié)構(gòu)域而并非此前有過報道的FReD結(jié)構(gòu)域。同時我們對D5結(jié)構(gòu)域中負責鈣離子結(jié)合的位點進行分析研究,通過細菌凝集、細菌結(jié)合和多糖結(jié)合的實驗驗證了三個可能的與鈣離子結(jié)合相關(guān)的位點(G53-G54-G55)。我們進一步將zITLN1和zITLN2的D5結(jié)構(gòu)域進行互換,構(gòu)建了ZITLN1-2D5和ZITLN2-1D5兩個嵌合體重組蛋白,對它們的凝集素功能進行研究,研究發(fā)現(xiàn)兩個嵌合體重組蛋白的凝集素功能都是與其所包含的D5結(jié)構(gòu)域的功能相一致,進一步說明D5是zITLN當中重要的糖識別功能域,D5結(jié)構(gòu)域決定了全長的糖結(jié)合能力。另外,我們利用ELISA技術(shù)檢測到zITLN1、ZITLN2和ZITLN3重組蛋白都可以與小牛乳鐵蛋白進行結(jié)合,但是zITLN1、zITLN2和zITLN3重組蛋白并不具備殺菌功能。以上對斑馬魚3個ITLN結(jié)合多糖從而結(jié)合細菌、凝集細菌的研究能夠加深我們對ITLN基因參與細菌感染引起的免疫防御機制的理解,而對于糖識別結(jié)構(gòu)域的研究初步探索了斑馬魚腸道凝集素糖識別的機制,為intelectin基因在其他病理和生理過程中的研究提供新的線索。而腸道凝集素作為一種與纖膠凝蛋白(Ficolin)序列相似度極高的蛋白,其糖識別結(jié)構(gòu)域卻是完全不同,這暗示其三級結(jié)構(gòu)的差異決定了其功能的不同,這也為我們將來的研究指明了方向,解析斑馬魚Intelectin的結(jié)構(gòu)進而更深入的了解其模式識別的機制。
[Abstract]:The role of the immune system is the most fundamental recognition of "self" and "non self" innate immunity is the first line of immune, the innate immune system is mainly composed of a series of genes encoding receptors to complete for the recognition of self and nonself, these receptors are known as pattern recognition receptors (PRRs). The common pathogenic molecular components, such as cell wall polysaccharides and double stranded RNA virus, can be recognized by the PRRs, and triggered a series of signaling pathways to the expression of immune related genes were regulated, so as to clear the pathogen. The effect of lectin (Lectin) can not only directly involved in immune defense. It is a pattern recognition molecule, plays an important role in the innate immune response in the gut. Lectin (Intelectin, ITLN) is a recently discovered a combination of beta galactose coagulation in furan, in a variety of Play an important role in the life activities, and many human diseases such as asthma, heart disease, enteritis, chronic obstructive emphysema and cancer have a certain relationship. The current research on intestinal lectin mainly in mammals, amphibian animal, teleost fish and ascidians, Wenchang. Although the ITLN sequence is highly conserved in different species. But they are in the expression pattern and function also has some differences. In view of the important function of ITLN in pattern recognition, the mechanism of pattern recognition is very important. Zebrafish as a model organism important, complete with the innate immune and acquired immune system, the immune system at the molecular and cellular level all mammals and a high degree of similarity. So we chose the intestinal lectin of zebrafish (zebrafish Intelectin zITLN) to study the pattern recognition mechanism. We first use the method of bioinformatics, the Intelectin zebrafish evolutionary status analysis, selected in three different branches of the zebrafish Intelectin:zITLN1, zITLN2 and zITLN3 were studied. We were cloned for the 3 zITLN, using real-time fluorescence quantitative PCR (Real-time PCR zITLN1) method of adult zebrafish the zITLN2 and zITLN3 distribution are explored, the experimental results show that the 3 zITLN in the eye, skin, intestine, gill, liver and tail were expressed, but the expression patterns and the expression is not the same: zITLN1 and zITLN2 mainly expressed in the intestine and liver expression is very low, but different zITLN3 with the first two, mainly expressed in the liver, but its expression in the intestine is relatively high. We also use Real-time method to detect the PCR of Staphylococcus aureus to zebra The regulation of fish Intelectin expression. Results showed that Staphylococcus aureus can cause zITLN1 up-regulated zITLN2 and zITLN3 expression, but not the same degree and pattern of zITLN1 and zITLN3 for Staphylococcus aureus stimulated more sensitive reaction, after injection of Staphylococcus aureus in 4 hours the expression. 8 hours to reach the highest value, while zITLN2 in 8 hours before the increase slowly, until 10 hours suddenly reached the peak. We used the Escherichia coli expression system for expression of 3 ITLN, and the expression products were purified. Bacterial agglutination test, fluorescence microscopy showed that the calcium ion, zITLN1 zITLN2, and the recombinant ZITLN3 protein can cause different degrees of FITC labeled bacteria agglutination, zITLN1 and zITLN3 as agglutination ability of Staphylococcus aureus is stronger than agglutination in Escherichia coli, and zITLN2 In two kinds of bacterial aggregation did not show significant differences in.Western blotting results showed that 3 ITLN can be combined with bacteria, zITLN1 and zITLN3 alloy of Staphylococcus aureus than with Escherichia coli, but for the two kinds of bacteria with ZITLN2 did not show significant difference.ELISA quantitative analysis results show that the zITLN1 and zITLN3 on the affinity of PGN is higher than LPS, and zITLN2 in polysaccharide combination has no obvious bias. To sum up, the zITLN2 binding capacity of polysaccharides was higher than that of zITLN1 and zITLN3. the results showed that zITLN1, zITLN2 and zITLN3 can identify the major component of the bacterial cell wall, which can be combined with bacteria and cause bacteria the agglutination. But their recognition and binding effect and mechanism of follow-up may be different. In order to further explore the carbohydrate recognition domain of intelectin gene within, we The construction of zITLN1-D5, zITLN2-D5 and ZITLN3-D5 three deletion mutants, by bacterial agglutination, bacterial binding and polysaccharide binding experiments for the first time that the amino acid sequence of C zITLN at the end of 100 or so in the zebrafish (D5) in which bacterial agglutination, bacterial binding and sugar binding function with no obvious differences in the length. It is responsible for the identification of polysaccharide ITLN is a D5 domain and the FReD domain had not previously reported. At the same time, we analyze the D5 domain responsible for calcium binding sites by bacterial agglutination, bacterial binding and polysaccharide binding experiments confirmed three possible and calcium binding sites related (G53-G54-G55) we will further the D5 domain of zITLN1 and zITLN2 are interchangeable, constructed the ZITLN1-2D5 and ZITLN2-1D5 two chimeric recombinant protein, to study their functions of lectins. Two chimeric recombinant protein lectin functions are consistent with the D5 domain contains the function, proves that D5 is an important function of zITLN carbohydrate recognition domain, D5 domain determines the length of the sugar binding ability. In addition, we use the ELISA technology to detect zITLN1, ZITLN2 and ZITLN3 recombinant protein can combined with calf lactoferrin but zITLN1, zITLN2 and zITLN3 recombinant protein does not possess the function of disinfection. More than 3 of zebrafish ITLN binding polysaccharides and combined with bacteria, bacteria can deepen our research on aggregation of ITLN genes involved in the immune defense mechanism caused by bacterial infection of the understanding, and the study of carbohydrate recognition domain of the preliminary exploration the mechanism of zebrafish intestinal lectin carbohydrate recognition, provide new clues for the study of intelectin gene in other physiological and pathological processes in the gut as a lectin. With fiber gel protein (Ficolin) sequences of high similarity of the protein, the carbohydrate recognition domain is completely different, suggesting that differences in its tertiary structure determines its function, it also pointed out the direction for our future research, structural analysis of zebrafish Intelectin and a deeper understanding of the mechanism of pattern recognition.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q78

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