【作者】 贾玉萍；

【导师】 王金星；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 甲壳动物集约化的水产养殖,特别是对虾的养殖,是水产养殖中增长最快的产品之一。尽管取得了巨大的成功,但是对虾养殖业仍面临着严重传染性疾病暴发流行的危险,这给对虾养殖业造成巨大的经济损失。自上世纪90年代以来,商业化的对虾养殖面临着严重的打击,病毒和弧菌引起的虾病的暴发流行,给养殖业造成了严重的经济损失。疾病的爆发常由三种因素的相互作用引起,即恶化的环境、弱抵抗力的宿主、侵略性的病原菌。甲壳动物没有获得性免疫;但具有先天性免疫包括酚氧化酶原系统激活引起的黑化作用,对外源物质的凝结、吞噬作用和包囊作用,抗菌作用和细胞凝集作用。抗菌肽通常是小的阳离子分子,广泛分布于整个生物王国,特别是对缺乏获得性免疫的生物是必须的。疾病的控制和生物安全目前被认为是影响对虾养殖业持续稳定发展的最关键因素。增强对虾的先天免疫可以提高对病原微生物感染的广谱抵抗力,激活或提高先天免疫系统能力的各种免疫增强药物的活性被普遍认为是一个用于提高养殖对虾的健康和抵抗流行病暴发很好的选择。免疫增强药物是那些能够刺激免疫反应和提高免疫系统抵抗感染和疾病能力的物质。如β-葡聚糖能够增强甲壳动物抵抗寄生虫、细菌和病毒的感染能力。来自对虾自身的抗菌肽应用于预防控制对虾疾病,即可以提高对虾的先天免疫力、抵抗病原微生物的入侵,又可以确保生物安全,因此研究对虾的抗菌肽,特别是具有双重功能的抗菌肽具有重要意义。抗菌肽在对虾体内行使功能不是独立完成的,需要免疫系统和免疫相关的信号转导途径的因子及其它因子的参与,因此研究免疫相关的信号转导机制中起作用的基因,了解对虾的信号转导通路在病原微生物入侵时在先天免疫中的作用具有重要意义,进而可以更好的了解对虾抵抗病原微生物的机制。目前的研究表明含有WAP（乳清酸蛋白）结构域的蛋白,具有蛋白酶抑制活性或抗菌活性。对虾同其它海洋无脊椎动物一样,免疫相关的信号转导机制目前研究的还比较少。本研究对中国明对虾含单一乳清酸性蛋白结构域的抗菌肽（Fc-SWD）基因、中国明对虾谷氨酰胺合成酶（Fc-GS）基因、中国明对虾Ras（Fc-Ras）基因进行了克隆鉴定,并对其蛋白功能进行了初步探讨,为进一步了解中国明对虾的免疫防御机制和免疫相关的信号转导机制提供理论依据。一、中国明对虾Fc-SWD的功能研究对虾依靠先天免疫系统生活于水生环境中并承受着各种压力,如细菌、真菌、病毒的入侵。抗菌肽的生成和释放是抵抗这些病原菌入侵的重要免疫反应。从中国明对虾的血细胞中克隆到了一个含单一WAP结构域的新型抗菌肽,命名为含单一乳清酸性蛋白结构域的抗菌肽（a single whey acid protein domaincontaining peptide,Fc-SWD）。该抗菌肽cDNA全长399bp,含有一个273bp的开放阅读框,编码90个氨基酸,包含24个氨基酸的信号肽和66个氨基酸的成熟肽。序列比较和进化树分析表明,对虾中已报道的含有WAP结构域的肽/蛋白分成3类:crustinⅠ、Ⅱ和SWD。Fc-SWD是SWD亚族的新成员。Northern blot分析表明Fc-SWD转录体在正常对虾血细胞中组成型表达,在感染的对虾血细胞中表达增加。但是在正常和感染对虾的心脏、肝胰腺、胃、鳃、肠、卵巢中未检测到表达。RT-PCR结果表明在正常和刺激的对虾血细胞、心脏、鳃中及感染对虾的胃中有Fc-SWD的表达。原位杂交结果表明Fc-SWD在对虾三种血细胞中均有表达,但是在其中的一类细胞中mRNA的表达信号最强:免疫组化结果也表明Fc-SWD蛋白在某一类细胞中有很强的表达信号:这些结果表明Fc-SWD在对虾某一类血细胞（可能是颗粒细胞）高表达而不是所有细胞中表达。Western blot分析表明Fc-SWD在血细胞和血淋巴中均有信号检出,且血细胞中的分子量大于血淋巴中的分子量,说明Fc-SWD在血细胞中合成且含有信号肽,当病原菌感染时以成熟肽分泌到血淋巴中发挥作用。成功构建了Fc-SWD基因的三种原核表达载体:原核重组表达载体Fc-SWD（含有信号肽的全长序列）,mFc-SWD（成熟肽）和Fc-WAPD（仅含有WAP结构域）,并对三种蛋白进行了诱导表达和纯化;利用牛津杯法和最小抑菌浓度的方法研究了纯化后三种重组蛋白的抗菌活性:利用生物化学和酶学方法分析了纯化后三种重组蛋白的抑制蛋白酶活性。结果表明重组蛋白Fc-SWD和mFc-SWD对革兰氏阳性菌、革兰氏阴性菌和真菌都有抑制作用,并且对枯草蛋白酶A和蛋白酶K有较强的抑制作用,对胰蛋白酶的抑制作用较弱;Fc-SWPD几乎没有抑菌作用,但是有抑制蛋白酶的活性,对枯草蛋白酶A和蛋白酶K的抑制作用强于对胰蛋白酶的抑制作用。这些研究结果表明Fc-SWD可能是对虾抵抗各种病原菌的免疫系统中重要的效应因子之一。Fc-SWD具有抗菌作用和抑制蛋白酶作用,是一种具有双重功能、具有广泛的开发前景和药用价值的生物活性肽。二、中国明对虾Fc-GS的功能分析谷氨酰胺合成酶（GS）是一个广泛分布的酶,以3种不同的蛋白形式存在于所有生物体中:GSⅠ型家族,主要在原核生物中发现:GSⅡ型家族,主要存在于真核生物中和少数细菌中;GSⅢ型家族主要在原核生物中发现。GS在免疫系统、神经系统、细胞增殖与分化、胚胎发育、大分子合成、围食膜基质的形成等方面发挥着重要作用。本试验成功克隆到了中国明对虾Fc-GS基因,其cDNA全长1376bp,含有一个1086bp的开放阅读框,编码361个氨基酸。编码蛋白的分子量约40.47 kDa、等电点为6.32。同源性比较和进化树分析表明中国明对虾Fc-GS基因属于GSⅡ家族。利用RT-PCR方法对细菌或病毒感染对虾的血细胞、心脏、肝胰腺、胃、鳃、肠和卵巢中Fc-GS的表达进行了分析:同时也对感染后不同时间段的肝胰腺、鳃和卵巢的表达情况进行了分析:结果表明病原菌感染的对虾中,Fc-GS的表达在基因水平上被高度调控。细菌和病毒感染对虾的胃和肠中的Fc-GS mRNA表达水平明显增加;病毒感染对虾血细胞、鳃和卵巢中Fc-GS mRNA水平显著上调,细菌感染的鳃中Fc-GS表达量增加。利用Western blot方法对正常、弧菌刺激和对虾白斑综合征病毒（WSSV）刺激对虾的肝胰腺、心脏、胃、肠、卵巢和肌肉组织中Fc-GS的蛋白质表达水平进行了分析,结果表明在检测的组织中均有表达,并且弧菌和WSSV刺激的胃、肠和卵巢中Fc-GS的表达明显增加。细菌和病毒感染的鳃、胃、肠和肌肉中Fc-GS蛋白也被诱导表达。成功构建了中国明对虾Fc-GS的原核表达载体,得到了重组蛋白,并对重组蛋白和对虾肌肉中Fc-GS进行了酶学分析。酶活性分析表明原核重组表达的重组中国明对虾Fc-GS合成酶活性最佳pH是6.0-7.5,最佳温度是37℃。细菌和病毒感染对虾肌肉中Fc-GS的合成酶活性与正常对虾的相比显著增加。与正常对虾相比,细菌或病毒感染的对虾肌肉中Fc-GS的合成酶活性增加了2倍。这些结果表明中国明对虾Fc-GS可能在抵抗病原微生物入侵,特别是病毒感染的免疫反应中起作用。基因和蛋白的上调作用可以作为检测对虾被病毒感染双重标准。该基因的研究对了解中国明对虾Fc-GS参与抗病毒免疫相关的作用机制提供了参考依据。为了解中国明对虾被病原菌感染后的健康状态提供了方法依据。三、中国明对虾Fc-Ras特性分析原癌基因Ras是一种多功能的细胞因子,该基因编码的蛋白是信号转导的重要组织者,直接参与细胞内的转导途径和影响其它细胞信号转导途径。Ras蛋白是一种小GTP结合蛋白,对细胞外信息的跨膜传递起着重要作用,被称为细胞信号网络传递中的“分子开关”。克隆到的中国明对虾Fc-Ras基因cDNA全长为1013bp,含有561bp的开放阅读框编码186个氨基酸。编码区含有3个EGF₁（EGF-like domain signature 1）（53-64,255-266,394-405）位点:1个2FE2S_FER₁（2Fe-2S ferredoxins,iron-sulfur binding region signature 1）（73-81）位点;编码蛋白的分子量约21kDa、等电点为6.37。用RT-PCR的方法分析Fc-Ras在正常、弧菌或WSSV刺激的对虾血细胞、心脏、肝胰腺、胃、鳃、肠、卵巢或精巢的表达模式。与正常对虾相比,弧菌刺激后的血细胞、肝胰腺、鳃、胃中Fc-Ras mRNA的表达明显增加;心脏、卵巢中的表达降低;刺激的肠中仅有微弱的增加。WSSV刺激对虾的血细胞、心脏、鳃、胃、肠、卵巢、精巢中Fc-Ras mRNA表达量明显增加,肝胰腺中增加不明显。用Western blot方法分析了中国明对虾Fc-Ras在不同组织中的表达变化:正常的胃、肠中没有Fc-Ras蛋白的表达,但是菌刺、毒刺胃和肠中Fc-Ras蛋白高表达;正常、菌刺、毒刺卵中均有Fc-Ras表达,且表达量增加。免疫组化的结果表明中国明对虾血细胞中有Fc-Ras的表达但是并不是所有的细胞都有很强的表达,而某一类细胞整体有很强的表达信号。WSSV刺激后的各组织中mRNA水平均有不同程度的增加,在检测的各组织中Fc-Ras蛋白的表达也增加,这些研究结果表明Fc-Ras可能参与对虾抗WSSV的免疫。对了解先天免疫相关的信号转导途径在病原微生物入侵对虾时所起的作用具有重要意义。结论:本研究结果表明Fc-SWD具有抗菌活性和抑制蛋白酶活性双重功能,是中国明对虾先天免疫中防御病原菌感染的重要抗菌肽之一。Fc-GS可能参与抵抗病原微生物的感染有关,特别是参与抵抗病毒的感染。Fc-Ras参与中国明对虾的抗病毒免疫,可能与中国明对虾先天免疫相关的信号转导途径有关。更多还原

【Abstract】 Intensive aquaculture of crustaceans,especially of penaeid shrimp,is one of the fastest growing sectors in aquaculture production.Despite its huge success,shrimp culture is facing severe outbreaks of infectious diseases.During the past few years, commercial shrimp farming has been severely hit due to epidemics associated with viruses and vibriosis,which have caused serious economic losses.Disease outbreaks are resulting from interactions of the three factors of a deteriorating environment,weakened hosts,and aggressive pathogens.Crustaceans do not have acquired immunity;instead they have an innate immune system,which includes melanisation by activation of the prophenoloxidase-activating system（proPO system）,a clotting process,phagocytosis,encapsulation’ of foreign material, antimicrobial action,and cell agglutination.The control of diseases and biological security have been regarded as the key factors which affect the continuous and stabilizing development of penaeid shrimp culture.Enhanced the innate immune shrimp can increase resistance to infection of broad-spectrum.Activated innate immune system or increased the variety of immune-enhancing drug activity is generally considered to be a good choice for prawn breeding to improve the health and resist epidemic outbreaks of disease. Immune-enhancing drugs are the material that can stimulate the immune response and enhancing the immune system to resist infection and disease.Such asβ- glucan,can enhance crustaceans resistance parasites,bacteria and virus infection capacity.Antibacterial peptide from their own shrimp for disease prevention and control can not only enhance the innate immune shrimp,resist the invasion of pathogenic microorganisms,but also to ensure biosafety.So the research of the shrimp antibacterial peptide,in particular,which has a dual function of antibacterial peptide is of great significance.Antibacterial peptide of shrimp in the exercise of functions is not an independent body to complete the required immune system and immune-related signal transduction pathway of participation and the participation of other factors common to complete the function.The study of genes related to immune-related signal transduction mechanism,understanding the shrimp signal transduction pathway in the invasion of pathogenic microorganisms in the role of innate immunity is of great significance,thereby better understanding of pathogenic microorganisms shrimp resistance mechanisms.The presence of WAP domains within proteins has been suggested to confer either proteinase inhibitor or antibacterial activity on these molecules and the mechanism of immuno-related signal transduction is not extensively studied in shrimp as well as in marine invertebrates.The Fc-SWD,GS and Ras cDNAs were cloned in the studies.The charaters and functions of the three genes could help us know about the inmmune defence mechanisms and the signal transduction mechanisms.1.A single whey acidic protein domain（SWD）-containing peptide from the fleshy prawn with antimicrobial and proteinase inhibition activitiesShrimp rely upon an innate immune system to live in marine environments under a variety of stresses,including bacterial,fungal,and viral challenges.The production and release of antimicrobial peptides are an important immune response to fight these pathogens.A gene for a new type of antimicrobial peptide that contains a single whey acidic protein domain（SWD） was cloned from the haemocytes of fleshy prawns （Fenneropenaeus chinensis） and this peptide is named Fc-SWD.The full-length cDNA of Fc-SWD was 399 bp and contained a single open reading frame（ORF） of 273 bp,encoding an Fc-SWD of 90 amino acids,including a putative 24-amino acid signal peptide,and a mature 66 residue peptide.Sequence comparison and phylogenetic analysis of SWDs and crustins in penaeid shrimp suggest that the whey acidic protein domain（WAP） containing peptides/proteins can be divided into three classes:crustinⅠ,Ⅱand SWD,and Fc-SWD is a new member in the SWD subgroup.Northern blot analysis demonstrated that the Fc-SWD transcripts were constitutively expressed in haemocytes of non-infected shrimp and increased in haemocytes of infected shrimp,but Fc-SWD mRNA was not detected in the hearts,hepatopancreas,stomachs,gills,intestines or ovarys of non-infected or infected shrimp.Reverse transcription polymerase chain reaction（RT-PCR） experiments showed that the Fc-SWD signal was detected in the haemocytes,hearts and gills of both unchallenged and challenged shrimp,and in stomach of challenged shrimp.In situ hybridization demonstrated that three cell types - hyaline,semi-granular, and granukar cell- in shrimp expressed Fc-SWD but only one of them has the strongest signals of expression.The cellular localization of the Fc-SWD protein in the haemocytes of the normal and the challenged shrimps was detected by immunocytochemistry.The peptide signal was predominantly located in the cytoplasm of some haemocytes,so the expression signal of Dc-SWD is probably in the cytoplasm of granular cells.Western blot analysis indicated that Fc-SWD protein was expressed in haemocytes and challenged haemolymphs,what’s more,the molecular weight of the band found in haemocute samples was larger than that detected in the challenged haemolymphs.So the Fc-SWD protein appeared to be synthesized in haemocytes with a signal peptide and then secreted to the haemolymph as a mature peptide when F. chinensis was infected by pathogens.Fc-SWD（for the full length peptide including the signal peptide）,mFc-SWD（for the mature peptide） and Fc-WAPD（for the single WAP domain） were expressed in Escherichia coli,respectively.Purified recombinant Fc-SWD and mFc-SWD have antimicrobial activities against gram-positive（G⁺） and gram-negative（G^-） bacteria, and fungi,as well as strong inhibitory activity against subtilisin A and protein K.The Fc-WAPD has anti-protease activity and low antimicrobial activity.These results suggest that Fc-SWD may be one of the key effectors in the shrimp immunity against various pathogens.Fc-SWD has antimicrobial and proteinase inhibition activities,and is a double function of antimicrobial peptides and broad prospects of bioactive peptides for development and medicinal value.2.Cloning and functional analysis of a glutamine synthetase in Chinese shrimp Fenneropenaeus chinensisGlutamine synthetase is a widely present in all organisms and distributed to 3 diffeent forms protein:GSⅠ-family,mainly found in prokaryotes;GSⅡ-family, mainly in eukaryotic Biology and in a small number of bacteria;GSⅢ-family found mainly in prokaryotes.GS plays an important role in the immune system,nervous system,cell differentiation and proliferation,embryonic development,the synthesis of molecules, peritrophic membrane to form the matrix,and so on.A glutamine synthetase cDNA（Fc-GS） was cloned from Chinese shrimp Fenneropenaeus chinensis.The full length cDNA of Fc-GS is 1376 bp with an open reading frame（ORF） of 1086 bp encoding a protein of 361 residues.Fc-GS has a calculated molecular mass of 40.47 kDa and pI of 6.32.Alignment of amino acid sequences and phylogenetic analysis indicated that Fc-GS belongs to GSⅡ-family.Expression of Fc-GS in haemocytes,heart,hepatopancreas,stomach,gill,intestine and ovary after bacterial- or white spot syndrome virus（WSSV）-challenged shrimps, and the time course expression of Fc-GS in hepatopancreas,gills and ovary of shrimps were investigated by RT-PCR.Our results showed that the Fe-GS was highly regulated in pathogen infected shrimp comparing to for all treatment.The Fc-GS mRNA level and protein concentration increased in the stomach and intestine in both bacterial and WSSV challenged shrimp.Expression of Fc-GS mRNA in haemocytes, gill,stomach and ovary was up-regulated after shrimp were injected with WSSV, while Fc-GS transcript was induced only in gill and stomach after bacterial infection.Protein expression of Fc-GS in hepatopancreas,heart,stomach,gill,ovary and muscles of the nomal shrimp,bacterial or white spot syndrome virus --challenged shrimps were analyzed by Western blot.The results indicated that Fc-GS was expressed in all tissues and increased in gill,stomach and ovary of bacteria-or WSSV-challenged shrimp.Fc-GS protein was also induced in the gill,stomach, muscle and intestine after bacterial or viral infection.Recombinant Fc-GS was expressed in Escherichia coli.The optimum pH for the recombinant Fc-GS（rFc-GS） was 6-7.5 and the optimum temperature was 37℃.The Fc-GS activity in muscles was increased significantly after bacterial or viral infection and the activity of Fc-GS enzyme in muscles was increased 2 folds in the shrimp infected with bacteria or WSSV compared to the uninfected shrimp.These results suggest that Fc-GS may play roles in immune responses against pathogen infection,especially viral infection.The Up-regulation of the gene and protein may be a biomarker for viral infection.The gene research provides a reference to understanding the Chinese shrimp glutamine synthetase involved in the immune-related signaling mechanism,and also provides a method basis to understanding the health state of Chinese shrimp after infected with bacteria or virus.3.Cloning and Characterization of Ras from the Chinese shrimp Fenneropenaeus chinensisProto-oncogene Ras is a multifunctional cytokine,the gene encoding the protein is an important signal transduction organizers,the cells directly involved in the pathway and other effects of cell signaling pathway.Ras protein is a small GTP-binding protein,the cell’s outer membrane transfer of information plays an important role in cell signaling network known as the transmission of the "molecular switch."The full length of the cDNA of Fc-Ras of F.chinensis was 1013 bp including a 561 bp open reading frame that encoding 186 amino acids.There are three EGF₁ （EGF-like domain signature 1）（53-64,255-266,394-405）,and one 2FE2S _FER₁（2Fe-2S ferredoxins,iron-sulfur binding region signature 1）（73-81） in open reading frame.Fc-Ras has a calculated molecular mass of 21 kDa and pI of 6.37.Haemocytes,heart,hepatopancreas,stomach,gills,intestine,ovary and spermary of normal and bacteria or WSSV challenged F.chinensis were selected to study the expression patterns of Fc-Ras with RT-PCR method.The data indicated the Fc-Ras gene expressed in all the selected tissues at different levels.There is a strong increased signal in Haemocytes,hepatopancreas,stomach,gills,and lower increase in intestine;but decreased in heart and ovary in bacteria-challenged shrimp.In the WSSV-challenged shrimp,there is a strong increased signal in Haemocytes,heart, stomach,gills,intestine,ovary and spermary,but no obviously increase in hepatopancreas.The results of western blot indicated that the Fc-Ras protein level was increased in stomach,intestine and ovary of bacteria-chanllenged and WSSV-challenged shrimp. Immunocytochemistry analysis of Fc-Ras in haemocytes indicated that the Fc-Ras strongly expressed in one type of shrimp haemocytes. It is of great significance to understand the role of innate immune-related signaling pathway when shrimp were in invasion by pathogenic microorganisms.In summary,a single whey acidic protein domain（SWD）-containing peptide has antimicrobial and proteinase inhibition activities.It is an important antimicrobial peptides for Fenneropenaeus chinensis innate immune defense.Fc-GS may be involved in the resistance of pathogenic micro-organisms,especially the resistance of virus.Fc-Ras may participate in the antiviral immunity and may be related to the signal transduction of Fenneropenaeus chinensis innate immunity.更多还原