【作者】 王妍；

【导师】 王晓梅；

【作者基本信息】 天津农学院 ， 水产养殖， 2015， 硕士

【摘要】 高密度、集约化的水产养殖使养殖对象频发各种细菌性及病毒性疾病,在病害防治的过程中,抗生素的使用不仅导致病原体产生耐药性、无法有效控制疾病的发生,而且药物残留还破坏了水环境的微生态平衡、严重威胁人类的食品安全。因此,急需研发新型、绿色的生物防治药物和饲料添加剂。抗菌肽具有广谱的抑菌活性和对病毒的杀伤作用,不易产生抗药性,因此成为首选的抗生素替代品。此外,加快对鱼类免疫防御机制的基础研究,也是有效防治病害发生,实现健康养殖及养殖业可持续发展的重要战略之一。革胡子鲶（Clarias gariepinus）生长快、抗病力强、可高密度养殖,并且具有加工优势,而在加工过程中有近50%的副产品产生。故本研究以高密度养殖、健康的革胡子鲶为试验对象,从其加工副产品中分离提取具有抑菌活性的抗菌蛋白/肽,挖掘其加工副产品的利用价值,为开发绿色的生物防治药物和饲料添加剂提供技术支持。此外,分析革胡子鲶不同组织器官中免疫因子基因（抗菌肽、溶菌酶和主要组织相容性复合体）的表达谱,为探究革胡子鲶的高抗病机理奠定基础。本文主要研究结果如下:1.革胡子鲶抗菌蛋白/肽的提取及体外活性分析以健康的革胡子鲶为研究材料,通过乙酸（Acetic acid,AA）和硫酸铵盐析（Ammonium sulfate,AS）两种方法从革胡子鲶加工副产品中分离提取具有抑菌活性的抗菌蛋白/肽。以获得量、抑菌效果和蛋白质分子种类作为衡量指标,建立抗菌蛋白/肽粗提物的分离方法。通过正交试验L₉（3⁴）得出乙酸提取法最优提取条件为:乙酸浓度为10%,提取时间为12 h,液料比（mL:g）为5:1。通过单因素试验得出硫酸铵分级盐析法以从饱和度60%开始,经过饱和度80%、100%的盐析法为优。综合考虑操作方便和抗菌蛋白/肽的稳定性,最终选择硫酸铵分级盐析法进行革胡子鲶抗菌蛋白/肽粗提物的分离。将饱和度100%硫酸铵盐析获得的抗菌蛋白/肽粗提物进行葡聚糖凝胶层析,对获得的峰2样本（ASP3-100%-b）进行SDS-聚丙烯凝胶电泳检测,电泳图谱上主要呈现相对分子质量为4.1KD的蛋白组份。对ASP3-100%-b样本进行抑菌活性和稳定性分析,杯碟法抑菌检测表明其对金黄色葡萄球菌、嗜水气单胞菌、温和气单胞菌和大肠杆菌均具有明显的抑制作用,抑菌圈直径分别为:8.34 mm、6.76 mm、9.27 mm和6.13 mm;微量稀释法测定其最小抑菌浓度分别为:105μg/mL、210μg/mL、210μg/mL和420μg/m L。采用微量反应板法进行稳定性分析,结果表明:经胃蛋白酶、胰蛋白酶及蛋白酶K处理后,对上述4种受试菌的抑菌率至少达到（56.8±0.1）%,表现出较强的抗蛋白酶水解的能力。该组份在80℃下保温1 h对上述4种菌的抑制率分别为:（92.6±0.3）%、（99.8±0.3）%、（90.6±1.0）%和（90.5±3.3）%;沸水浴180 min处理后抑菌率分别为:（55.7±0.2）%、（65.2±0.5）%、（66.8±0.5）%和（54.7±0.2）%;经高压灭菌处理后抑菌率分别为:（76.6±1.7）%、（88.7±0.7）%、（67.5±3.8%）和（61.2±2.4）%,表现出较好的热稳定性;经过反复冻融10次该组份对上述4种受试菌的抑菌率分别为:（100±4.2）%、（66.1±2.7）%、（95.3±3.0）%、（70.7±4.4）%,显示出较好的反复冻融稳定性;此外该组份在较高（pH=11）或较低（pH=3）的pH值下,对上述4种受试菌的抑菌率均保持在（72.3±1.2）%以上,具有较好的pH稳定性。2.革胡子鲶免疫因子的基因表达分析应用实时荧光定量PCR（qPCR）技术,分析了3种免疫因子基因:抗菌肽（AP）、溶菌酶（LYS）和主要组织相容性复合体Ⅰ（MHCⅠ）,在健康、高密度养殖革胡子鲶不同组织和器官中的相对表达谱。首先,根据同源克隆的方法,应用Primer 5.0软件,共设计17对扩增AP、LYS和MHCⅠ基因片段的引物。筛选获得3对引物(APF₁₁-APR₁₁、LYSF₃-LYSR₃和MHCF₁-MHCR₃),进一步对其进行qPCR评估,熔解曲线和标准曲线的结果显示:引物具有良好的特异性和扩增效率。在此基础上,分析了革胡子鲶鳃、鳃上器官、脑、心脏、头肾、肾脏、肝胰脏、脾脏、胃、肠道、肌肉、皮肤和鳍13种组织中AP、LYS和MHCⅠ基因mRNA水平的表达差异。以鳃的表达量作为基准,数据统计分析结果显示:AP基因在脾脏中相对表达量最高,是鳃表达量的3.3倍,与其他12种组织的表达量差异显著（P<0.05）;LYS基因在胃中的表达水平最高,达到鳃表达量的11.8倍,显著高于其他12种组织的表达水平（P<0.05）;MHCⅠ基因在脾脏和肠道中表达量相对较高,在脑、头肾、肝胰脏及胃中表达量相对较低,但13种组织间MHCⅠ基因的相对表达量差异不显著（P>0.05）。更多还原

【Abstract】 High stocking density and intensive aquaculture mode cause the outbreaks of bacterial and viral disease in farmed fish.In the process of the disease prevention and treatment,the antibiotics are being used as prophylactic and therapeutic agents,which not only result in the development of resistant strains of bacterial pathogen,but also destroy the micro-ecological balance of the water and are harmful to consumer’s health because of the inevitable residues of antibiotics in water and in the end product.Therefore,it has stimulated enormous efforts to develop alternative antimicrobial agents and the feed additive to prevent and control disease.As the antimicrobial proteins/peptides display broad-spectrum antimicrobial activity,virus cytotoxicity,and do not induce bacterial resistance,they may be possibly exploited as promising candidates instead of antibiotics.In addition,the research on immune defense mechanism of fish is one of the important strategies in preventing the occurrence of disease and realizing healthy aquaculture and sustainable development.Clarias gariepinus is one of the most important cultured fish owing to its fast growth,high disease resistance and high stocking-density capacities.The fish also has the processing advantages and there are 50%of the by-products during the process.In this paper,bioactive antibacterial proteins/peptides were isolated from the by-products of healthy C.gariepinus reared at high stocking density.The aims of the study were to exploit utility value of the by-products of C.gariepinus and establish an applicable method for development of alternative antimicrobial agents and new feed additive.Moreover,gene expression of immune factors（antimicrobial peptides,lysozyme and major histocomp atibility complexⅠ）in different tissues and organs of C.gariepinus were analyzed,which lay a foundation for understanding the high disease resistance mechanism of this fish.The main research results are as follows:1.Extraction and in vitro activity of antibacterial proteins/peptides of C.gariepinusAcetic acid（AA）and ammonium sulfate（AS）were respectively used for extracting crude antibacterial proteins/peptides from C.gariepinus by-products and extraction process were optimized with the yield,antibacterial activity and variety of the crude proteins/peptides extracts as test indices.The extraction condition with acetic acid optimized by using orthogonal design L9（3⁴）was 10%acetic acid,12h extraction and 5:1 liquid to solid ratio.With the method of single factor experiment,the ammonium sulfate fractionation methods were screened.The result showed that the crude proteins/peptides were precipitated starting at60%saturation followed by 80%and 100%saturation,which was the better.Taking into account easy operation and the stability of antibacterial proteins/peptides,we eventually use the method of AS fractionation for isolating crude antibacterial proteins/peptides from by-products of C.gariepinus.Crude antibacterial proteins/peptides obtained at 100%saturation AS were subject to gel filtration chromatography of Sephadex G-75 and G-50.The protein profile of the sample（ASP3-100%-b）pooled from absorption peaks 2 by SDS-PAGE gel showed that the molecular weights of major components were about 4.1KD.The antibacterial activity and stability of ASP3-100%-b were assayed.The results were following:The ASP3-100%-b exhibited antibacterial activity against Staphyloccus aureus,Aeromonas hydrophila,Aeromonas sobria and Escherichia coli in Oxford cup bacteriostatic test,the diameter of the inhibition zone was 8.34,6.76,9.27 and 6.13 mm,respectively.The minimal inhibitory concentration against the above bacteria,measured by means of 2-fold microdilution method,respectively was 105,210,210 and 420μg/mL.The inhibitory rate of ASP3-100%-b against these bacteria cited above reached（56.8±0.1）%or more after the sample was treated with pepsin,trypsin and proteinase K,and the stability under the proteinase K treatment was the best.The inhibitory rate of ASP3-100%-b against these bacteria was（92.6±0.3）%,（99.8±0.3）%,（90.6±1.0）%and（90.5±3.3）%,respectively,or（55.7±0.2）%,（65.2±0.5）%,（66.8±0.5）%and（54.7±0.2）%.,respectively,after the sample was incubated at 80℃for 1 h or100℃for 3 h.After high pressure sterilization treatment,the inhibitory rate still reached（76.6±1.7）%,（88.7±0.7）%,（67.5±3.8）%and（61.2±2.4）%respectively.The inhibitory rate of ASP3-100%-b,underwent freezing and thawing 10 times,respectively was（100±0.4）%,（66.1±2.7）%,（95.3±3.0）%and（70.7±0.4）%.The inhibitory rate of ASP3-100%-b were unaffected by pH at 11 and 3.These results indicated that the sample,ASP3-100%-b exhibited good stability.2.Analysis of gene expression of immune factors in C.gariepinusRelative expression profiles of the genes of antimicrobial peptides（AP）,lysozyme（LYS）and major histocompatibility complexⅠ（MHCⅠ）in different tissues and organs of healthy C.gariepinus were detected by using real-time PCR（qPCR）.Firstly,by means of homology cloning method,17 pairs of primer in total were designed using Primer 5.0 software for amplifying the fragments of AP,LYS and MHCⅠ,respectively.Three primer pairs of them,namely,APF₁₁-APR₁₁,LYSF₃-LYSR₃ and MHCF₁-MHCR₃ could respectively be used in analyzing the relative expression of AP,LYS and MHCⅠdue to that these primers displayed good specificity according to their melting curves and good amplification efficiency based on their amplified standard curves in qPCR assessment.Expression of the AP,LYS and MHCⅠgenes,in different organs including gill,suprabranchial organ,brain,heart,head-kidney,kidney,hepatopancreas,spleen,stomach,intestines,muscles,skin and fins of the fish were assayed by qPCR.Relative mRNA level of these genes cited above were normalized against their mRNA level in gill.The results showed that AP gene was highest expression in spleen（3.3 fold compared to that in gill）and there were significant differences in AP gene mRNA levels between spleen and other 12 organs（P<0.05）.The highest gene expression of LYS gene was observed in stomach which was 11.8 fold to that in gill,and the differences were significant compared to the other 12 tissues（P<0.05）.However,MHCⅠgene were highly expressed in spleen and intestine and lower expressed in brain,head-kidney,hepatopancreas and stomach,there were no significant differences in MHCⅠgene mRNA levels between any two organs（P>0.05）.更多还原