【作者】 李新苍；

【导师】 王金星；

【作者基本信息】 山东大学 ， 动物学， 2010， 博士

【摘要】 克氏原螯虾(红沼泽螯虾,也称淡水小龙虾,属节肢动物门,甲壳纲,十足目,蜊蛄科,原螯虾属)是一种在实验室内研究无脊椎动物先天免疫的良好模型,同时也是一种重要的经济动物。利用其开展先天免疫研究,特别是开展抗甲壳动物主要病原(白斑综合征病毒和弧菌)免疫机理研究,必将进一步丰富甲壳类动物的先天免疫理论,同时也可为甲壳动物疾病的防治提供重要的理论参考。白斑综合征病毒(WSSV)是对虾的主要病原之一,同时也是其他甲壳动物如淡水小龙虾、蟹及龙虾等的重要病原。为研究WSSV对小龙虾的感染规律,并为进一步开展小龙虾先天免疫研究提供必要的数据,本研究首先建立了以实时定量PCR为基础的WSSV定量方法。研究结果表明,使用该方法定量WSSV具有快速、灵敏及准确的特点,很好的解决了下一步WSSV感染实验感染剂量的问题。以前研究表明淡水小龙虾重要的免疫相关基因主要存在于血细胞和肝胰腺中。为了深入地研究小龙虾的先天免疫机制及抗WSSV机理,我们首先用一定量的WSSV感染小龙虾,感染96 h后,取含血细胞丰富的鳃和肝胰腺组织混合物提取mRNA,用于构建小龙虾的cDNA文库。对构建的cDNA文库进行测序、在线比对及序列整理,从中鉴定了四个具有全长cDNA序列的Kazal型丝氨酸蛋白酶抑制因子,分别命名为hcPcSPI1、hcPcSPI2、hpPcSPI3及hpPcSPI4。为研究这四个抑制因子的组织分布及进化关系,我们首先采用半定量RT-PCR方法检测这四个基因在RNA水平的组织分布。随后对这四个抑制因子做了全长及单结构域的同源性比较,并进一步分析与其它物种抑制因子的进化关系。研究结果显示hcPcSPI1和hcPcSPI2主要来源于血细胞,hpPcSPI3和hpPcSPI4主要分布于肝胰腺和心组织；同源性比较显示,这四个抑制因子同源性为42.47%,其中来源于肝胰腺的两个抑制因子的同源性为56.67%,而来源于血细胞的hcPcSPI1与hcPcSPI2同源性只有23.73%。进化树分析显示,与来源于对虾肝胰腺的七个抑制因子形成一个大的分支不同,hpPcSPI3和hpPcSPI4形成一个较小的分支。表明这两个抑制因子从进化关系上看与其它肝胰腺型抑制因子距离较远;而它们两个之间同源性较高,进化相对保守。另外,来源于甲壳动物血细胞的这些抑制因子形成了另一个较大的分支,但节点值较低,表明来源于甲壳动物血细胞的抑制因子进化较快。为研究这四个抑制因子是否参与抗WSSV的免疫反应,并进一步检测是否参与抗细菌的免疫反应,采用实时定量RT-PCR的方法检测了在WSSV或细菌刺激条件下四个抑制因子在转录水平上的变化规律。结果显示：在受到WSSV刺激时hcPcSPI2和hpPcSPI3表达量上调,hcPcSPI1的表达先下调而后逐渐恢复,而hpPcSPI4表达量没有变化；在受到革兰氏阴性细菌刺激后,四个抑制因子表达量均上调。以上结果表明：hcPcSPI1、hcPcSPI2和hpPcSPI3以不同的方式参与小龙虾的抗WSSV免疫反应；同时这四个抑制因子还可能均参与小龙虾的抗革兰氏阴性细菌的免疫反应。存在于甲壳动物血淋巴中的Kazal型丝氨酸蛋白酶抑制因子被认为参与调控宿主的免疫防御反应或对抗入侵病原微生物分泌的蛋白酶。因此,我们对来源于血细胞的两个抑制因子做了进一步的研究。对hcPcSPI1序列分析结果表明：该抑制因子具有三个Kazal结构域,其活性位点的氨基酸分别是L、L和E。该蛋白具有信号肽,并且在其第一与第二个结构域之间还含有一个RGD基序。在蛋白水平上hcPcSPI1主要存在于血细胞中,同时在其他组织如心、鳃和肠中也有少量分布。蛋白水平的进一步分析显示其在受到大肠杆菌刺激后表达上调,表明其可能参与抗大肠杆菌的免疫反应。为进一步研究该抑制因子的生物学及生理功能,首先对hcPcSPI1及其三个结构域分别进行原核表达及纯化。蛋白酶活性抑制实验表明：这四个蛋白对枯草蛋白酶A及蛋白酶K都具有抑制活性,而对胰凝乳蛋白酶和胰蛋白酶没有抑制活性。重组的hcPcSPI1能结合大肠杆菌、克雷伯氏菌、枯草芽孢杆菌、苏云金芽孢杆菌、金黄色葡萄球菌、白色念珠球菌和毕赤酵母；进一步研究显示只有结构域1可以结合大肠杆菌和金黄色葡萄球菌,另外两个结构域没有细菌结合活性。另外,重组的hcPcSPI1还能抑制枯草芽孢杆菌和苏云金芽孢杆菌的生长,结构域2和3对这种抑制活性起作用。以上结果表明hcPcSPI1在小龙虾的先天免疫反应中起着重要的作用。hcPcSPI2具有两个Kazal结构域,其活性位点的氨基酸分别是R和S。Westernblot结果显示hcPcSPI2只存在于半颗粒细胞中,而不存在于透明细胞和颗粒细胞中。hcPcSPI2在受到鳗弧菌刺激后表达量升高,并且可以释放到血淋巴中发挥作用。为了研究该抑制因子的抑制功能,对该抑制因子进行原核表达、纯化,检测其对五种蛋白酶的抑制活性。结果显示其对枯草蛋白酶和胰蛋白酶有一定的抑制活性。而进一步的酶抑制动力学研究表明该抑制因子为竞争性抑制因子,并且其Ki值分别为0.057μM和3.729μM。细菌生长抑制实验表明,该抑制因子对枯草芽孢杆菌及苏云金芽孢杆菌的生长均有抑制作用,其MIC50分别为30.4μM和25.0μM。这些研究结果表明hcPcSPI2可能在小龙虾的抗细菌免疫反应中发挥重要的作用。更多还原

【Abstract】 It’s well known that the crayfish has become an excellent animal model to study the innate immunity of the invertebrate, as well as an important economic animal. The studies on the innate immune responses of crayfish, especially on immune defense against the main crustacean-pathogens such as white spot syndrome virus (WSSV) or Vibrio anguillarum, will further enrich the knowledge of the innate immunity of crustaceans and provide important theory for controling the infectious diseases.WSSV is one of the main pathogens of shrimp, and it’s also the pathogen of the other crustaceans such as crayfish, crab, lobster, etc. In order to investigate the distribution of WSSV in the different tissues of the infected crayfish and further provide the required data for the following studies of innate immunity of crayfish, a quantitative PCR to quantify the amount of WSSV was developed. The results indicated that this method to quantify the number of WSSV was rapid, sensitive, and precise. This developed method well satisfied the need of the following WSSV infection experiment.For crayfish, the important immunity-related proteins are synthesized mainly in hemocytes and hepatopancreas, and some of them are further secreted into the circulating hemolymph to function. To investigate the mechanism of the defense against WSSV and other pathogens, the healthy crayfish were challenged by WSSV first. At 96h after challenge the gills full of hemocytes and hepatopanceas were collected to isolate the mRNA, and then the mRNA was transcribed into cDNA to construct the cDNA library. After randomly sequencing, four Kazal-type inhibitors, namely hcPcSPI1, hcPcSPI2, hpPcSPI3, and hpPcSPI4, with full-length cDNA sequence were identified from this cDNA library.To examine the tissue distribution of these four inhibitors, four pairs of primers for these four inhibitors were designed. Using the semi-quantitative RT-PCR the tissue distribution of these four inhibitors at mRNA level were detected. The results demonstrated that hcPcSPI1 and hcPcSPI2 mainly existed in hemocytes while hpPcSPI3 and hpPcSPI4 mainly distributed in hearts and hepatopancreas. Homology comparison indicated that the identity of these four inhibitors was 42.47%, hpPcSPI3 shared higher 56.67%identify with hpPcSPI4, and hcPcSPI1 only share 23.73% identity with hcPcSPI2. The phylogenectic tree constructed using the inhibitors of the crustaceans showed that hpPcSPI3 and hpPcSPI4, both derived from the hepatopancreas, are different from other hepatopancreas type inhibitors of shrimp, formed a small group. This indicates that hpPcSPI3 and hpPcSPI4 have a relatively distant evolutionary relationship with other inhibitors from hepatopancreas. In addition, the inhibitors from hemocytes seem hard to be grouped into one meaningful cluster since the Bootstrap values are very low, which reveals that these hemocyte type inhibitors, including hcPcSPI1 and hcPcSPI2, evolve rapidly.In order to examine whether these four inhibitors were involved in the immune defense against WSSV, as well as defense against V. anguillarum for hpPcSPI3 and hpPcSPI4, real-time RT-PCR was used to detect the expression profiles of these inhibitors after WSSV or V. anguillarum challenge. The results indicated that after WSSV challenge, hcPcSPI2 and hpPcSPI3 were up-regulated, hcPcSPI1 first decreased and then gradually recovered, while there was no obvious change for hpPcSPI4. It’s possible that hcPcSPI1, hcPcSPI2, and hpPcSPI3 participate in the immune defense against WSSV, and hpPcSPI3 and hpPcSPI4 were also involved in the anti-V.anguillarum immune response.In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this paper, we further analyzed two Kazal-type serine proteinase inhibitors derived from hemocytes, namely hcPcSPI1 and hcPcSPI2, from the crayfish (Procambarus clarkia).We find that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099). In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not a-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPIl was also found to possess bacteriostatic activity against B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish.The serine proteinase inhibitor hcPcSPI2 is composed of a putative signal peptide, and two tandem Kazal-rype domains with the domain PI residues L and E, respectively. The temporal expression profile of this inhibitor was studied with quantitative real-time PCR and the results suggest that hcPcSPI2 is likely to be involved in anti-F. anguillarum immune response. Western blot demonstrates that hcPcSPI2 only exists in semigranular cells. Besides, after V. anguillarum challenge, the hcPcSPI2 protein could also be detected in cell-free hemolymph. Subsequently, the biochemical characteristics and bacteriostatic activity of hcPcSPI2 were assayed. The results indicate that hcPcSPI2 shows weak inhibitory activity against subtilisin A and trypsin, and may trigger bacteriostatic activity towards B. subtilis and B. thuringiensis, with MIC50 of 30.4 and 25.0μM, respectively. These studies reveal that hcPcSPI2 may also play an important role in the anti-bacterial immunity of the crayfish.更多还原