本文關(guān)鍵詞： 斑馬魚 腸道凝集素 模式識(shí)別 糖識(shí)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(shí)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(duì)于“自己”和“非己”的識(shí)別主要是由一系列基因編碼受體來(lái)完成的,這些受體被稱為模式識(shí)別受體(PRRs)。病原分子共有的成分,比如細(xì)胞壁的多糖以及RNA病毒的雙鏈等,能夠被PRRs識(shí)別,并引發(fā)一系列的信號(hào)通路來(lái)對(duì)各種免疫相關(guān)基因的表達(dá)進(jìn)行調(diào)控,從而達(dá)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(shí)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(lái)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(dòng)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(dòng)物、兩棲動(dòng)物、硬骨魚、文昌魚和海鞘當(dāng)中。雖然不同物種的ITLN序列高度保守,但是它們?cè)诒磉_(dá)模式和功能上也存在一定差異。鑒于ITLN在模式識(shí)別中的重要功能,研究其模式識(shí)別的機(jī)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(shí)其免疫系統(tǒng)在分子和細(xì)胞水平上都與哺乳動(dòng)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(lái)研究其模式識(shí)別的機(jī)制。我們首先利用生物信息學(xué)的技術(shù)手段,對(duì)斑馬魚Intelectin的進(jìn)化地位進(jìn)行分析,選擇了處于三個(gè)不同分支上的斑馬魚Intelectin:zITLN1、zITLN2和zITLN3來(lái)進(jìn)行研究。我們對(duì)這3個(gè)zITLN進(jìn)行了克隆,利用實(shí)時(shí)熒光定量PCR(Real-time PCR)的方法對(duì)成體斑馬魚的zITLN1、zITLN2和zITLN3的組織分布進(jìn)行了探索,實(shí)驗(yàn)結(jié)果表明3個(gè)zITLN在腸道、眼球、皮膚、鰓、肝臟以及尾鰭中均有表達(dá),但表達(dá)模式及表達(dá)量不盡相同：zITLN1和zITLN2主要集中在腸道中表達(dá),肝臟中表達(dá)量很低,而zITLN3與前兩者不同,主要集中在肝臟中表達(dá),當(dāng)然其在腸道中的表達(dá)量也相對(duì)較高。我們同樣利用Real-time PCR的方法檢測(cè)了金黃色葡萄球菌對(duì)斑馬魚Intelectin表達(dá)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(dá)上調(diào),但是程度與模式不盡相同：zITLN1和zITLN3對(duì)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(shí)就出現(xiàn)了表達(dá)的上調(diào),8小時(shí)達(dá)到最高值,而zITLN2在前8小時(shí)中的上調(diào)很緩慢,直到10小時(shí)突然達(dá)到峰值。我們利用大腸桿菌表達(dá)系統(tǒng)對(duì)3種ITLN進(jìn)行了表達(dá),并對(duì)其表達(dá)產(chǎn)物進(jìn)行純化。細(xì)菌凝集實(shí)驗(yàn)中,熒光鏡檢顯示,鈣離子存在時(shí),zITLN1、zITLN2和ZITLN3重組蛋白均可以不同程度的引起FITC標(biāo)記的細(xì)菌的凝集,zITLN1和zITLN3一樣凝集金黃色葡萄球菌的能力要強(qiáng)于凝集大腸桿菌的能力,而zITLN2對(duì)于兩種細(xì)菌的凝集卻沒(méi)有表現(xiàn)出明顯差異。Western blotting結(jié)果顯示3個(gè)ITLN都可以結(jié)合細(xì)菌,zITLN1和zITLN3一樣結(jié)合金黃色葡萄球菌的能力要強(qiáng)于結(jié)合大腸桿菌的能力,但是ZITLN2對(duì)于兩種細(xì)菌的結(jié)合沒(méi)有表現(xiàn)出明顯差異。ELISA定量分析的結(jié)果表明zITLN1和zITLN3對(duì)PGN的親和性要高于LPS,而zITLN2在多糖結(jié)合上沒(méi)有明顯的偏向性。總起來(lái)說(shuō),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(shí)別細(xì)菌細(xì)胞壁的主要成分,從而能結(jié)合細(xì)菌并引發(fā)細(xì)菌的凝集。但是它們的識(shí)別和結(jié)合效應(yīng)以及后續(xù)的作用機(jī)制可能不同。為了進(jìn)一步探究intelectin基因內(nèi)在的糖識(shí)別結(jié)構(gòu)域,我們構(gòu)建了zITLN1-D5、zITLN2-D5和ZITLN3-D5三個(gè)缺失突變體,通過(guò)細(xì)菌凝集、細(xì)菌結(jié)合和多糖結(jié)合的實(shí)驗(yàn)在斑馬魚當(dāng)中首次發(fā)現(xiàn)zITLN序列C末端100個(gè)左右的氨基酸(D5)在細(xì)菌凝集、細(xì)菌結(jié)合和糖結(jié)合方面的功能與其全長(zhǎng)并無(wú)明顯差異。說(shuō)明ITLN中負(fù)責(zé)多糖識(shí)別的是D5結(jié)構(gòu)域而并非此前有過(guò)報(bào)道的FReD結(jié)構(gòu)域。同時(shí)我們對(duì)D5結(jié)構(gòu)域中負(fù)責(zé)鈣離子結(jié)合的位點(diǎn)進(jìn)行分析研究,通過(guò)細(xì)菌凝集、細(xì)菌結(jié)合和多糖結(jié)合的實(shí)驗(yàn)驗(yàn)證了三個(gè)可能的與鈣離子結(jié)合相關(guān)的位點(diǎn)(G53-G54-G55)。我們進(jìn)一步將zITLN1和zITLN2的D5結(jié)構(gòu)域進(jìn)行互換,構(gòu)建了ZITLN1-2D5和ZITLN2-1D5兩個(gè)嵌合體重組蛋白,對(duì)它們的凝集素功能進(jìn)行研究,研究發(fā)現(xiàn)兩個(gè)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(jìn)一步說(shuō)明D5是zITLN當(dāng)中重要的糖識(shí)別功能域,D5結(jié)構(gòu)域決定了全長(zhǎng)的糖結(jié)合能力。另外,我們利用ELISA技術(shù)檢測(cè)到zITLN1、ZITLN2和ZITLN3重組蛋白都可以與小牛乳鐵蛋白進(jìn)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(duì)斑馬魚3個(gè)ITLN結(jié)合多糖從而結(jié)合細(xì)菌、凝集細(xì)菌的研究能夠加深我們對(duì)ITLN基因參與細(xì)菌感染引起的免疫防御機(jī)制的理解,而對(duì)于糖識(shí)別結(jié)構(gòu)域的研究初步探索了斑馬魚腸道凝集素糖識(shí)別的機(jī)制,為intelectin基因在其他病理和生理過(guò)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(shí)別結(jié)構(gòu)域卻是完全不同,這暗示其三級(jí)結(jié)構(gòu)的差異決定了其功能的不同,這也為我們將來(lái)的研究指明了方向,解析斑馬魚Intelectin的結(jié)構(gòu)進(jìn)而更深入的了解其模式識(shí)別的機(jī)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(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78

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