【作者】 张莹；

【导师】 宋林生；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2010， 博士

【摘要】 甲壳动物在水产养殖业中占据十分重要的地位,深入开展免疫防御机制的相关研究,探索免疫防治新途径,是实现其健康养殖的可靠保障。本研究通过RACE技术获得了凡纳滨对虾的整连因子LvIntegrin,C型凝集素LvLec,以及中华绒螯蟹的细胞因子抑制因子EsSOCS2和抗脂多糖因子EsALF-2的cDNA全长序列。通过RT-PCR法研究了它们对不同微生物刺激的响应,并对功能结构域原核重组蛋白进行了相关活性测定。同时检测了CpG ODNs注射处理后中华绒螯蟹的部分免疫指标和受WSSV感染的凡纳滨对虾的存活率、病毒拷贝数及抗病毒相关因子的变化,初步获得以下结果。凡纳滨对虾LvIntegrin和LvLec cDNA全长为2621和618 bp,分别具有典型的特征结构域β-integrin亚基和CRD。LvIntegrin在受到鳗弧菌侵染时,mRNA表达水平均显著提高。重组LvIntegrin具有Ca2+依赖的大肠杆菌、鳗弧菌、溶壁微球菌和毕赤酵母的凝集活性,同时具有促进人成纤维细胞增殖的作用。LvIntegrin基因干扰后感染鳗弧菌的对虾死亡率升高,抗体阻断LvIntegrin蛋白后,感染鳗弧菌的对虾中酚氧化物酶原基因的表达受到抑制。重组的LvLec具有Ca2+依赖的大肠杆菌凝集活性,该活性可被甘露糖抑制,提示该蛋白属于甘露糖结合型的C型凝集素。中华绒螯蟹EsSOCS2和EsALF-2 cDNA全长分别为2535和724 bp,EsSOCS2含有典型的ESS、SH2和SOCS box三个结构域。EsSOCS2和EsALF-2在各检测组织中呈组成型表达,且分别在鳗弧菌、藤黄微球菌和鳗弧菌、毕赤酵母的诱导下,表达量显著增加。重组的EsALF-2具有抑制鳗弧菌和毕赤酵母生长的作用,而对革兰氏阳性菌不起作用。合成CpG ODNs处理中华绒螯蟹和凡纳滨对虾,发现0.1μg的CpG ODNs可抑制ROS的产生,促进酚氧化物酶和溶菌酶活性增强,并诱导抗菌肽Crustin的mRNA表达。经CpG ODNs预处理的对虾感染WSSV后,存活率显著高于对照组,且对虾体内病毒拷贝数显著降低,同时对虾血淋巴细胞中ROS水平和Dicer、STAT的mRNA表达量显著增加,而Argonaute表达水平未发现显著变化。提示CpG ODNs通过间接抑制WSSV病毒的复制,诱导对虾防御WSSV的免疫保护作用。结果表明,LvIntegrin、LvLec、EsSOCS2和EsALF-2是虾蟹类重要的免疫防御因子,均参与了凡纳滨对虾和中华绒螯蟹的免疫防御反应。CpG ODNs能激活虾蟹类固有免疫防御体系,在抵御WSSV侵染过程中起重要保护作用,可作为免疫增强剂用于虾蟹类养殖动物的免疫防治。该研究结果对深入了解海洋无脊椎动物免疫防御机制,发展免疫防治方法和途径具有理论意义和应用价值。更多还原

【Abstract】 Crustaceans in aquaculture occupy a very important position, and the knowledge of immune defense mechanisms as well as the development of novel immunostimulants is helpful for disease control in crustacean aquaculture. In the present study, the genes of LvIntegrin and LvLec from Litopenaeus vannamei, EsSOCS2 and EsALF-2 from Eriocheir sinensis were cloned by rapid amplification of cDNA ends (RACE) techniques. Their expression profiles in crustaceans challenged by several microorganisms were detected by RT-PCR technique, and their functions were examined by assaying their recombinant protein expressed in Escherichia coli BL21(DE3)-plysS. The immune protective effects of CpG ODNs for crustaceans against pathogenic microorganisms were also evaluated.The full-length cDNA of LvIntegrin and LvLec was 2621 and 618 bp, with typical domain ofβ-integrin subunit and CRD respectively. The mRNA expression of LvIntegrin was up-regulated in haemocytes of shrimps challenged by Listonella anguillarum. Recombinant LvIntegrin displayed agglutinative activities against E. coli, L. anguillarum, Micrococcus lysodeikticus and Pichia pastoris and promoted the proliferation of human fibroblasts. Recombinant LvLec represented agglutinative activity only aginst E. coli JM109. The agglutinative activities of LvIntegrin and LvLec were both of Ca2+ dependent. The mortality rates were significantly increased in LvIntegrin-RNAi-shrimps treated with L. anguillarum, and the EsproPO gene was inhibited in LvIntegrin-blocking-shrimps challenged by L. anguillarum.The full-length cDNA of EsSOCS2 and EsALF-2 was 2535 and 724 bp respectively, and EsSOCS2 contained three typical domains of ESS, SH2 and SOCS box. The mRNA expression of EsSOCS2 and EsALF-2 was both up-regulated in crabs challenged by the microorganisms. The recombinant EsALF-2 displayed antibacterial activity against L. anguillarum and P. pastoris, but no antibacterial activity against Gram-positive bacteria.The cellular and humoral immune responses were detected to evaluate the immune effects of CpG ODNs on innate immunity of crabs. The survival rates, WSSV copy numbers and several anti-virus factors were employed to evaluate the immune protective roles of CpG-C in shrimps against WSSV. 0.1μg of CpG-C could inhibit the ROS generation, enhance the activities of PO and lysozyme, up-regulate mRNA expression of Crustin. These results collectively suggested that CpG-C could activate innate immune responses of Chinese mitten crab, and might be used as a novel immunostimulant for disease control in E. sinensis. There were higher survival proportion, lower WSSV copy numbers, and higher mRNA expression of Dicer and STAT in CpG-ODNs-pretreatment shrimps than that in control shrimps after WSSV infection. The ROS levels in CpG-ODNs-pretreatment shrimps post secondary stimulation of PBS were significantly higher than those post WSSV infection. These results together demonstrated that CpG-ODNs induced partial protective immunity in shrimps against WSSV via intermediation of virus replication indirectly and could be used as a potential candidate in the development of therapeutic agents for disease control of WSSV in L. vannamei.The results together indicated that LvIntegrin, LvLec, EsSOCS2 and EsALF-2 were crucial immune factors in crustaceans and all involved in the immune defense responses. CpG ODNs could activate the innate immunity of crustaceans and played important roles in protecting the shrimps against WSSV. CpG ODNs as immunostimulants could be used for immune prevention and disease control in crustacean species. The results were helpful for better understanding immune defense mechanisms and developing novel immunostimulants for disease control in marine invertebrates.更多还原