【作者】 盛金良；

【导师】 陈创夫；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 脂多糖(lipopolysaccharide LPS)是革兰氏阴性细菌胞壁外膜中的一种生物活性成分,在革兰氏阴性细菌感染的发病机制中起十分重要的作用,能够刺激机体免疫系统,诱导炎症反应,而且作用于抗原提呈细胞,参与诱导抗感染的特异性免疫应答,是早期免疫应答反应的最强刺激剂。本项研究旨在构建LPS诱导的绵羊肺泡巨噬细胞cDNA文库,为绵羊免疫相关功能基因研究提供基础平台;监测免疫相关因子(细胞因子、TLR受体和防御素)基因对LPS刺激的应答反应规律,探讨其参与机体抗感染免疫的分子机制;通过基因工程表达相关功能基因并鉴定其生物学功能,为开发免疫佐剂和治疗性药物奠定基础。目前,绵羊各种组织类型的cDNA文库数量还很少,可为绵羊基因组研究提供的遗传标记很有限,导致绵羊基因组物理图谱研究较其它家畜如牛、猪等研究相对滞后。绵羊功能基因的研究主要集中在经济性状相关基因,对抗感染免疫相关的因子研究很不充分,绵羊细胞因子、toll样受体和防御素的表达分布、与病原感染之间的关系以及自身之间相互调节的联系都还不清楚,其表达调控的分子机制对研究疾病的病理过程具有重要意义,有必要进行深入的研究和探讨。本研究通过气管灌流法从绵羊肺脏分离培养原代肺泡巨噬细胞,利用台盼蓝拒染试验和鸡红细胞吞噬试验对其进行形态学观察和鉴定,运用SMART技术构建LPS诱导的绵羊肺泡巨噬细胞全长cDNA文库,随机挑选255个克隆进行序列测定,通过BLAST/n/x对序列进行比对分析,利用Sequin软件提交新发现EST或基因;应用PCR和RT-PCR技术扩增绵羊细胞因子(IL-1β,IL-8,GM-CSF,IFN-γ)、TLR受体家族、防御素sBD1和内标(G3PDH,α-Tubulin)基因,对其进行克隆和序列分析,以含有目标基因的质粒为模板建立荧光定量PCR检测方法,动态监测LPS诱导绵羊肺泡巨噬细胞过程中细胞因子、toll样受体和防御素的应答反应规律;应用RT-PCR和荧光定量PCR方法检测绵羊防御素sBD1在绵羊不同发育阶段的表达分布及表达差异。建立沙门氏菌感染绵羊消化道模型,检测防御素sBD1和IL-8对沙门氏菌感染的应答反应,对其抗感染生物学功能进行研究;构建重组绵羊GM-CSF表达质粒电击转入酵母细胞,通过甲醇诱导绵羊GM-CSF分泌表达。构建原核表达载体在大肠杆菌中融合表达绵羊防御素sBD1,并对其进行抗菌活性检测。初步获得以下结果:(1)构建了脂多糖LPS诱导的绵羊肺泡巨噬细胞全长cDNA文库,其库容量达到1.3×106,重组率为95.7%,符合cDNA文库建库标准;从255个随机克隆中发现新EST或基因45个并提交GenBank,获得登录号EU366470-EU366473,EU366475- EU366492, EU366494-EU366497,EU370534-EU370552,未知基因6个;(2)系统地阐明了在LPS刺激0～48h过程中,绵羊细胞因子(IL-1β,IL-8,GM-CSF,IFN-γ)、toll样受体家族和防御素的应答规律,即IL-1β在LPS刺激后6h达到最高值,IL-8,GM-CSF在诱导后4-12h表达水平最高,IFN-γmRNA水平高峰期在16h以后。toll样受体1、2、4、6、7、8、9、10均在肺泡巨噬细胞有表达,而toll样受体3、5则未见表达,TLR2、4在诱导后20min就达到高峰,且在整个诱导过程中维持较高水平;(3)防御素sBD1在绵羊肺泡巨噬细胞中没有表达,其主要在呼吸道和消化道组织中表达,而且是持续表达的,其在不同发育阶段消化道表达水平高低为羔羊>成年羊>胎羊。在感染沙门氏菌实验中IL-8明显升高,而防御素sBD1表达反而有所降低;(4)在毕赤酵母中诱导表达绵羊GM-CSF,其表达量占总蛋白17%;在大肠杆菌中融合表达了绵羊防御素sBD1,但未表现出抗菌活性。以上结果表明,本研究成功地构建了脂多糖LPS诱导的绵羊肺泡巨噬细胞cDNA文库,该文库为国内首个绵羊肺泡巨噬细胞cDNA文库,从对文库克隆的序列分析来看,新的EST和未知功能的基因约占20%,丰富了绵羊基因组物理图谱的遗传标记。另外,该文库是革兰氏阴性菌主要抗原组分脂多糖刺激巨噬细胞cDNA文库,为免疫相关的功能基因研究提供了平台;绵羊几种主要细胞因子(IL-1β,IL-8,GM-CSF,IFN-γ)对脂多糖LPS刺激表现出时间和表达量的差异为阐明疾病病理状态的分子机制提供了科学依据,toll样受体3、5和防御素sBD1在脂多糖刺激肺泡巨噬细胞中并不表达,说明肺泡巨噬细胞可能并不是其表达部位,toll样受体2、4对脂多糖LPS刺激的敏感性很高,说明它们参与早期的免疫应答反应,在革兰氏阴性菌感染中发挥重要作用;防御素sBD1在粘膜系统中广泛分布,并且持续表达,表明其在粘膜天然免疫中一定发挥着重要作用,但其对沙门氏菌感染没有反应,可能是由于其并不直接参与抵抗细菌感染,而与其它类型病原感染有关,而IL-8明显参与了消化道对沙门氏菌的抗感染免疫;绵羊GM-CSF参与早期的免疫应答,并且在疫苗佐剂和治疗性药物表现出良好应用前景,本研究在酵母中表达绵羊GM-CSF为进行开发和应用提供材料;防御素sBD1在粘膜系统广泛分布,但其生物学功能却不清楚,通过大肠杆菌融合表达绵羊防御素sBD1,为其生物学功能研究奠定基础。更多还原

【Abstract】 Lipopolysaccharide (LPS) is a predominant glycolipid of the outer membrane of gram-negative bacteria and plays a key role in gram-negative bacteria infectious disease.It can stimulate monocytes, macrophages, and neutrophils to produce cytokines; increases the expression of cell-adhesion molecules and induces the secretion of proinflammatory mediators.The aim of the study was to construct full-length cDNA library from LPS induced ovine alveolar macrophage and clone some of immune-related genes such as cytokines,toll-like receptors and defensin,detect the dynamics of these genes expression during LPS stimulation, clarify the molecular mechanism of cytokines,toll-like receptors(TLRs)and defensin participating in immune response. Through genetic engineering to express immune-related genes and identify its biological function, lay the foundation of the development for immune adjuvant and therapeutic drugs.At present, The number of sheep cDNA library in various cell types is seldom, and provide very limited genetic markers for the sheep genomic research, resulting in physical map of sheep genome is lagging behind than other domestic animals such as cattle, pigs. Sheep genomic function studies focused on traits of economic-related genes, however ,The immune-related factors research are still far from full. The mechanism of regulation on sheep cytokines(interleukin [IL]-1β, IL-8, granulocyte-macrophage colony stimulating factor [GM-CSF], and interferon [IFN]-γ), toll-like receptors and defensin is still not clear, but it is of great significance for elucidating pathologic process and is necessary to study in-depth.Ovine primary alveolar macrophages were cultured in vitro and identified with trypan blue exclusion and phagocytic experiment, The SMART technology was used to construct a full-length cDNA library from LPS induced ovine alveolar macrophages. Random of 255 clones of cDNA library were sequenced and align with BLAST/n and BLAST/x,the new expressed sequence tags(ESTs) were annotated and submitted to GenBank. Some of immue-related genes such as cytokines(IL-1β,IL-8,GM-CSF,IFN-γ),TLRs ,defensin and housekeeping genes(G3PDH,α-Tubulin)were cloned and sequenced with molecular biologic methods.The mRNA level of these genes response to LPS stimulated alveolar macrophage were investigated by using real-time RT-PCR. The distribution and variance of sheep beta-defensin1(sBD1) in developmental stages was determined by RT-PCR and real-time PCR.The response of sBD1 and IL-8 in Salmonella infective models were detected by real-time PCR.The recombinant sheep GM-CSF expression vector were constructed and transferred into yeast by electric shocks, and expressed secretroyly with methanol induction. Sheep beta-defensin1 were expressed in fusion in E.coli.Preliminary results as following: (1) cDNA library from LPS induced sheep alveolar macrophage was constructed, the capacity was 1.6×106 pfu/ml and recombinant rate reached 95.7%.45 new ESTs or genes from 255 effective EST sequences were submit to GenBank,the accession numbers were EU366470-EU366473, EU366475-EU366492, EU366494-EU366497,EU370534-EU370552, and discovered 6 unknown EST sequences.(2) The regular pattern of several cytokines(IL-1β,IL-8,GM-CSF,IFN-γ) and TLR family response to LPS stimuli were elucidated systematically. The mRNA expression of IL-1β,IL-8,GM-CSF were peaked between 4 and 12 h, the exception was IFN-γmRNA whose expression peaked around 16 h post-stimulation.The expression of TLR1,2,4,6,7,8,9,10 were detected exception that of TLR3,5.Further, TLR2 ,4 mRNA expression rapidly increased post-stimulation and peaked at 20 min post-stimulation and this high level was maintained throughout the procedure.(3)sheep alveolar macrophage was not target cell for sBD1 expression ,It was expressed consistently in the tissue of digestive and respiratory tract, the expression level in digestive tract was neonatal> adult>fetal sheep. The decreased change in expression of sBD1 was detected in digestive tract during Salmonella infection while IL-8 was Significant upregulation under this conditions.(4) Sheep GM-CSF was secreted expression in picha yeast and accouted for 17% of total protein; sBD1 were expressed in E.coli, But did not show antibacterial activity.The above results showed that we have firstly constructed a cDNA library of sheep alveolar macrophage induced with LPS,it provide a platform for the function of immune-related genes,the new EST accoutting for 20% enriched the genetic markers of sheep genome physical map;The regular pattern of the cytokines(IL-1β,IL-8,GM-CSF,IFN-γ) offer the scientific basis for elucidating the molecular mechanism of pathogenesis.Alveolar macrophage is not target cell for the expression of TLR3,5 and sBD1 while TLR2,4 are sensitive for LPS stimulation ,TLR2,4 plays an important role in response to gram-negative bacteria infection;The expression of sBD1 exclusively and consistently in mucous system show that it plays an important role in innate immunity,there was no response to the samonella infection may be due to its not directly involved in the resistance of bacterial infection but related with other type of pathogens.The IL-8 significantly participated in Anti-infective immunity for salmonella infection;Sheep GM-CSF involved in the early stage immune response,and shown a good prospect for development of adjuvant vaccine and therapeutic drug, so we expressed sheep GM-CSF in yeast for the preparation of its development and application; sBD1 in mucous system widely expressed but its biological function is unclear, sheep defensin-1 expressed in E.coli offere the opportunity to research its biologic function in vitro.更多还原