斑马鱼intelectin基因zITLN1、zITLN2、zITLN3的克隆与功能研究

发布时间：2018-02-10 13:53

本文关键词： 斑马鱼肠道凝集素模式识别糖识别结构域先天免疫　出处：《山东大学》2016年博士论文　论文类型：学位论文

【摘要】：免疫系统最根本的作用是识别“自己”和“非己”,先天性免疫是免疫的第一道防线,先天性免疫系统对于“自己”和“非己”的识别主要是由一系列基因编码受体来完成的,这些受体被称为模式识别受体(PRRs)。病原分子共有的成分,比如细胞壁的多糖以及RNA病毒的双链等,能够被PRRs识别,并引发一系列的信号通路来对各种免疫相关基因的表达进行调控,从而达到清除病原体的效果。凝集素(Lectin)不仅能够直接参与免疫防御,更是一种重要的模式识别分子,在先天性免疫应答中发挥重要作用。肠道凝集素(Intelectin, ITLN)是近年来新发现的一种能够结合β-呋喃半乳糖的凝集素,在多种生命活动中发挥重要作用,并且与人类的很多疾病如哮喘、心脏病、肠炎、慢性阻塞性肺气肿甚至癌症都有一定关系。目前关于肠道凝集素的研究主要集中在哺乳动物、两栖动物、硬骨鱼、文昌鱼和海鞘当中。虽然不同物种的ITLN序列高度保守,但是它们在表达模式和功能上也存在一定差异。鉴于ITLN在模式识别中的重要功能,研究其模式识别的机制显得尤为重要。斑马鱼作为一种重要的模式生物,具有较为完整的先天免疫和后天免疫系统,同时其免疫系统在分子和细胞水平上都与哺乳动物相似度很高。所以我们选择了斑马鱼的肠道凝集素(zebrafish Intelectin, zITLN)来研究其模式识别的机制。我们首先利用生物信息学的技术手段,对斑马鱼Intelectin的进化地位进行分析,选择了处于三个不同分支上的斑马鱼Intelectin:zITLN1、zITLN2和zITLN3来进行研究。我们对这3个zITLN进行了克隆,利用实时荧光定量PCR(Real-time PCR)的方法对成体斑马鱼的zITLN1、zITLN2和zITLN3的组织分布进行了探索,实验结果表明3个zITLN在肠道、眼球、皮肤、鳃、肝脏以及尾鳍中均有表达,但表达模式及表达量不尽相同：zITLN1和zITLN2主要集中在肠道中表达,肝脏中表达量很低,而zITLN3与前两者不同,主要集中在肝脏中表达,当然其在肠道中的表达量也相对较高。我们同样利用Real-time PCR的方法检测了金黄色葡萄球菌对斑马鱼Intelectin表达的调控,结果表明金黄色葡萄球菌可以引起zITLN1、zITLN2和zITLN3的表达上调,但是程度与模式不尽相同：zITLN1和zITLN3对于金黄色葡萄球菌的刺激反应更为灵敏,在注射金黄色葡萄球菌之后的4小时就出现了表达的上调,8小时达到最高值,而zITLN2在前8小时中的上调很缓慢,直到10小时突然达到峰值。我们利用大肠杆菌表达系统对3种ITLN进行了表达,并对其表达产物进行纯化。细菌凝集实验中,荧光镜检显示,钙离子存在时,zITLN1、zITLN2和ZITLN3重组蛋白均可以不同程度的引起FITC标记的细菌的凝集,zITLN1和zITLN3一样凝集金黄色葡萄球菌的能力要强于凝集大肠杆菌的能力,而zITLN2对于两种细菌的凝集却没有表现出明显差异。Western blotting结果显示3个ITLN都可以结合细菌,zITLN1和zITLN3一样结合金黄色葡萄球菌的能力要强于结合大肠杆菌的能力,但是ZITLN2对于两种细菌的结合没有表现出明显差异。ELISA定量分析的结果表明zITLN1和zITLN3对PGN的亲和性要高于LPS,而zITLN2在多糖结合上没有明显的偏向性。总起来说,zITLN2结合多糖的能力要高于zITLN1和zITLN3。上述结果表明：zITLN1、zITLN2和zITLN3可以识别细菌细胞壁的主要成分,从而能结合细菌并引发细菌的凝集。但是它们的识别和结合效应以及后续的作用机制可能不同。为了进一步探究intelectin基因内在的糖识别结构域,我们构建了zITLN1-D5、zITLN2-D5和ZITLN3-D5三个缺失突变体,通过细菌凝集、细菌结合和多糖结合的实验在斑马鱼当中首次发现zITLN序列C末端100个左右的氨基酸(D5)在细菌凝集、细菌结合和糖结合方面的功能与其全长并无明显差异。说明ITLN中负责多糖识别的是D5结构域而并非此前有过报道的FReD结构域。同时我们对D5结构域中负责钙离子结合的位点进行分析研究,通过细菌凝集、细菌结合和多糖结合的实验验证了三个可能的与钙离子结合相关的位点(G53-G54-G55)。我们进一步将zITLN1和zITLN2的D5结构域进行互换,构建了ZITLN1-2D5和ZITLN2-1D5两个嵌合体重组蛋白,对它们的凝集素功能进行研究,研究发现两个嵌合体重组蛋白的凝集素功能都是与其所包含的D5结构域的功能相一致,进一步说明D5是zITLN当中重要的糖识别功能域,D5结构域决定了全长的糖结合能力。另外,我们利用ELISA技术检测到zITLN1、ZITLN2和ZITLN3重组蛋白都可以与小牛乳铁蛋白进行结合,但是zITLN1、zITLN2和zITLN3重组蛋白并不具备杀菌功能。以上对斑马鱼3个ITLN结合多糖从而结合细菌、凝集细菌的研究能够加深我们对ITLN基因参与细菌感染引起的免疫防御机制的理解,而对于糖识别结构域的研究初步探索了斑马鱼肠道凝集素糖识别的机制,为intelectin基因在其他病理和生理过程中的研究提供新的线索。而肠道凝集素作为一种与纤胶凝蛋白(Ficolin)序列相似度极高的蛋白,其糖识别结构域却是完全不同,这暗示其三级结构的差异决定了其功能的不同,这也为我们将来的研究指明了方向,解析斑马鱼Intelectin的结构进而更深入的了解其模式识别的机制。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

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