【作者】 吴宗凡；

【导师】 王高学；

【作者基本信息】 西北农林科技大学 ， 水生生物学， 2012， 硕士

【摘要】 近年来，高密度、集约化的工厂化养殖模式为水产养殖带来了巨大的经济效益。然而，这种养殖方式往往会给鱼类造成较差的生存环境，使各种传染病的爆发率大大提高，造成大量的经济损失，制约着水产养殖业进一步可持续发展。免疫增强剂的使用，能够增强机体的非特异性免疫，达到广泛预防传染性疾病的效果，是水产养殖中控制和防止疾病发生行之有效的办法。本实验室前期研究发现，分离于温泉底泥样品的嗜热菌株厌氧芽孢杆菌SX-4（Anoxybacillus flavithermus SX-4）的发酵产物能够提高鲤鱼的非特异性免疫。本研究在此基础之上，采用硅胶柱层析、Sephadex LH-20凝胶过滤、重结晶等现代分离技术，从厌氧芽孢杆菌SX-4的发酵产物中分离单体化合物，并以草鱼肾细胞系（CIK细胞）为实验模型，研究分离得到的单体化合物对草鱼免疫相关基因表达的影响，评估其非特异免疫效果，获得如下结果：1．将厌氧芽孢杆菌SX-4接种于普通肉汤大量发酵后，经离心、大孔树脂DM130动态吸附、甲醇洗脱获得洗脱液，进一步浓缩、制备浸膏，拌入等量的硅胶搅拌均匀、干燥并研磨制成上样样品。样品经第一级硅胶柱层析分离，TLC检验合并相似流份为8个组分，即Fr.A-H。组分Fr. D和Fr. G再经第二级硅胶柱层析、Sephadex LH-20凝胶、重结晶等分离和纯化，最终获得单体化合物A和B。运用波谱学手段并结合理化性质鉴定：化合物A为对羟基苯乙酰胺（p-hydroxyphenylacetylamide），化合物B为环（甘氨酸-脯氨酸）二肽（cyclo-(Gly-L-Pro)）。2．将对羟基苯乙酰胺和环（甘-脯）二肽按照1μg/mL、10μg/mL和100μg/mL的浓度分别加入CIK细胞培养基中，在培养的2h、8h及24h分别取样，采用qRT-PCR技术分析样品中免疫相关基因MyD88、IL-1β、TNF-α、typeⅠ-IFN和IL-8的表达。结果发现：培养2h后，2种代谢产物均上调CIK细胞中MyD88的mRNA转录水平，并使之达到峰值，随后，MyD88的表达水平有所回落，但仍显著高于对照组（p<0.01）；在8h及24h，与空白对照组相比，IL-1β、TNF-α和typeⅠ-IFN3种细胞因子的基因表达量均显著性上调（p<0.05）。3．采用MTT法研究2种代谢产物对CIK活力的影响，评估其细胞毒性。结果显示：在实验浓度范围内，2种化合物在72h内对CIK细胞均未表现出毒性效应，而且，环（甘-脯）二肽在处理细胞48h后，CIK细胞的相对活力显著增加（p<0.05）。综上所述，从嗜热的厌氧芽孢杆菌SX-4的发酵产物中分离得到的代谢物对羟基苯乙酰胺和环（甘-脯）二肽，在离体的情况下能够诱导草鱼MyD88、 IL-1β、TNF-α和typeⅠ-IFN几种免疫相关基因的表达，并对CIK细胞无毒，具有开发成为新型免疫增强剂的潜力。更多还原

【Abstract】 Annual global aquaculture production has more than tripled within the span of15years,from16.8million tons in1990to52.9million tons in2005. Despite of the huge success, suchhigh production of aquaculture involves the intensive system of management practices, whichrenders the fish highly susceptible to different pathogens, facilitates transmissions ofinfectious pathogens and frequently induces outbreaks of emerging diseases. Application ofvarious immunostimulants to activate or boost the innate immune system has been widelyaccepted as a good strategy to improve health of cultured fish and fight epidemics.In our previous study, it has been demonstrated that the fermentation products ofthermophilic Anoxybacillus flavithermus SX-4, isolated from a sludge sample of Dongda hotspring (Shaanxi, China), was capable of increasing the non-specific immune parameters incarp (Cyprinus carpio) such as bactericidal activity and phagocytic activity by peripheralblood leucocytes. The present study isolated two purified compounds from the cell-freecultures of A. flavithermus SX-4, by column chromatography on silica gel and SephadexLH-20prior to recrystallization. Chemical structure of the isolated compounds wascharacterized by spectral analysis (nuclear magnetic resonance and mass spectrometry). Forthe purpose of evaluating the immunostimulatory activity of the two compounds, qRT-PCRwas used to investigate their effects on immune-related gene expression in grass carp(Ctenopharyngodon idella) kidney (CIK) cells. This study also examined the changes in cellviability induced by the isolated compounds and LPS. The resulsts of this study aresummarized as follows:1. A. flavithermus SX-4was inoculated in nutrient broth for7days on a reciprocal shakerwater bath at55℃. The combined culture broth (100L) was chromatographed onmacroporous resin DM130and eluted with methanol. The eluents were concentrated undervacuum by a rotary evaporator at65℃yielding92.0g of methanol extract. Part of methanolextract (70.0g) was put through a silica gel column (silica gel:100–200mesh) and elutedwith a solvent mixture of petroleum ether/ethyl acetate and ethyl acetate/methanol affording423fractions (500mL each). Fractions showing similar chromatograms (analyzed by TLC)were combined to yield8new fractions A–H. Fr. D and Fr. G were further separated and purified by column chromatography on silica gel and Sephadex LH-20prior torecrystallization, eventually leading to the isolation of two compounds (A and B). Based onthe physical and chemical properties and their spectral properties, compounds A and B wereidentified as p-hydroxyphenylacetylamide and cyclo-(Gly-L-Pro), respectively.2. In order to evaluate the immunostimulatory activity of the two isolated compounds,qRT-PCR was used to investigate their effects on gene expressions of MyD88and fourcytokines (IL-1β, TNF-α, IL-8and typeⅠ-IFN) in CIK cells. Expression profiling of theselected immune-related genes in cells stimulated with known immunostimulatory agent LPSwas also examined. The obtained results revealed that both isolated compounds, as did LPS,augmented the transcribing level of MyD88, an important adaptor molecule in toll-likereceptor (TLR) signaling pathway, as early as2h exposure. These compounds also inducedgene expression of cytokines in CIK cells such as IL-1β, TNF-α and typeⅠ-IFN.3. A MTT assay was employed to measure changes in cell viability, to analyze whetherthese compounds are toxic to the cells. The results demonstrated that in the case ofconcentrations remained below100μg/mL, none of the test compounds is toxic to the CIKcells at72h post-treatment. Moreover, cyclo-(Gly-L-Pro) and LPS could increase proliferationof the CIK cells.In summary, the present study demonstrates the immunostimulatory properties of the twometabolites (p-hydroxyphenylacetylamide and cyclo-(Gly-L-Pro)) from thermophilic A.flavithermus SX-4in CIK cells, and highlights the potential of using both metabolites asimmunostimulants in fish aquaculture.更多还原