【作者】 姜国建；

【导师】 周名江； 于仁诚；

【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， 2004， 博士

【摘要】 近年来，中国迅速发展的海水养殖业面临着病害和养殖环境退化等诸多问题的困扰，其中对虾养殖业受到白斑综合症病毒（WSSV）的冲击很大。本文针对这一问题，系统研究了对虾在白斑综合症病毒感染和环境因子胁迫下先天性免疫因子的动态变化过程，筛选出了能够准确反映对虾健康状况的免疫因子，进而将其运用到对虾抵抗 WSSV 的机制和环境胁迫对对虾健康状况的影响的研究中。 在对现有文献进行全面综述的基础上，我们通过实验系统比较了中国对虾和日本对虾在摄食感染 WSSV 后的死亡率、免疫学因子和感染病毒的变化情况,以及在氨氮胁迫下对虾感染 WSSV 过程中免疫学因子的变化情况。根据各种免疫学因子的变化情况，从中筛选出与 WSSV 感染直接相关的免疫学因子，并优选出一氧化氮合成酶（NOS）作为反映对虾抗病力的重要指标，它直接反映出了两种对虾对白斑综合症病毒抵抗能力的差异，应当是导致其对 WSSV 敏感性差异的主要原因之一。其水平也受氨氮胁迫的影响。 在此基础上，我们建立了对虾血细胞中诱导型一氧化氮合成酶（iNOS）的鉴定方法—硝基四氮唑蓝（NBT）还原法和血细胞形态法以及酶活力的测定方法—L-瓜氨酸分析法和亚硝酸盐分析法并优化了鉴定和测量条件，结果显示: 对虾血细胞在 4oC 下与 L-精氨酸和 LPS 孵育 8h 以及当 L-精氨酸浓度为 2.5mmol l-1,LPS 浓度为 100μg ml-1,Ca2+浓度为 2.5mmol l-1 时，iNOS 活力的测定结果最佳。 我们通过NBT还原法和血细胞形态法在中国对虾和日本对虾血细胞中鉴定到了iNOS，并通过亚硝酸盐法和L-瓜氨酸法对比研究了感染WSSV后这两种对虾血细胞中iNOS的变化情况,结果显示：在WSSV感染初期，WSSV可以诱导中国对虾和日本对虾产生iNOS，且日本对虾比中国对虾的iNOS更容易被LPS所诱导。此后随着病毒的增殖，iNOS活性显著降低，对虾也趋于死亡。iNOS活力的差异可能是日本对虾较中国对虾对WSSV的抵抗力强的原因之一。 本文在系统比较两种对虾感染WSSV病毒过程中抗病力指标变化的基础上，优选出与WSSV感染直接相关的抗病力指标,并首次对对虾的一氧化氮合成酶进行了系统的研究，所得到的结果为深入了解对虾对白班综合症病毒的抗病力机制,并建立和发展相应的病害防御措施提供了科学依据。更多还原

【Abstract】 The rapid development of the mariculture industry in China is now facingthe problems such as diseases and degradation of mariculture environment, amongwhich the shrimp-culture industry is particularly impacted by the disease of white spotsyndrome virus (WSSV). Focusing on this topic, we studied systematically thevariation of immune parameters of Chinese shrimp Fenneropenaeus chinensis andKuruma prawn Marsupenaeus japonicus after infection of WSSV by oral route, withattention of comparing the immunocompetence of the two species to WSSV infection.The immune parameters were also evaluated in Kuruma prawn M. japonicus under thestress of ammonia-N and WSSV infection. Based on these works, we discriminatedthe immune parameters closely related to WSSV infection from all the parametersused, and found nitric oxide synthase (NOS) a reliable parameter for indicatingdisease resistance ability to WSSV infection. Based on these findings, we developed nitro blue tetrazolium (NBT) method andcellular morphological approaches to identify the inducible NOS (iNOS) in thehaemocytes of Kuruma prawn M. japonicus and Chinese shrimp F. chinensis. Theconditions for NBT method were modified to make them suitable for the analysis ofiNOS in shrimp blood cells. Results showed that the optimal concentrations ofL-arginine, lipopolysaccharide (LPS) and Ca2+ for NBT method are 2.5mmol l-1,100μg ml-1 and 2.5mmol l-1, respectively. Meanwhile, two methods for iNOS activityassay based on the analysis of nitrite and L-citrulline concentrations were also appliedto study iNOS activity in the haemocytes of shrimp. The results showed that anincubation time of 8 hours is most suitable for the analysis of iNOS enzyme activity.The four methods could identify iNOS in shrimp haemocytes and analyze the activityeffectively, thus provide a good way for the further study of role of iNOS in innate 2<WP=12>对虾抗白斑综合症病毒的免疫学研究immunology of shrimps. Furthermore, the iNOS was identified in the haemocytes of Chinese shrimp F.chinensis and Kuruma prawn M. japonicus, and the methods developed wereemployed to evaluate their immunocompetence to WSSV infection in shrimps. Thevariations of iNOS activity in both penaeid species post-challenge of WSSV wereassayed with the methods of L-citrulline analysis and nitrite analysis. The resultshowed that iNOS activity in WSSV infected Chinese shrimp F. chinensis andKuruma prawn M. japonicus increased at 12 and 48 hours post-inoculum, butdecreased sharply after 36 and 72 hours post-inoculum respectively. LPS inducibleNOS activity also decreased significantly with the time elapsed in WSSV infectedshrimps. Results of WSSV detection with the Dig-DNA Probe dot blot hybridizationtest showed that WSSV of the two penaeid species couldn’t be detected until iNOSstarted decreasing. It can be concluded that WSSV infection can stimulate the iNOSsynthesis in shrimps. However, the enzyme activity decreased sharply after theproliferation of virus. Therefore, iNOS can be considered as a valuable index forindicating the health status of Chinese shrimp F. chinensis and Kuruma prawn M.japonicus. In comparison with Chinese shrimp F. chinensis, the iNOS in thehaemocytes of Kuruma prawn M. japonicus are more inclined to be induced by LPSafter WSSV infection at early stage of infection, which might account for therelatively longer surviving time of M. japonicus after WSSV infection. Based on the findings in the comparative study of immune parameters betweenChinese shrimp Fenneropenaeus chinensis and Kuruma prawn Marsupenaeusjaponicus, this thesis firstly carry on the researches of iNOS activity in shrimps, andfind it a reliable parameter for indicating the disease resistance ability in shrimps. Theresults here could offer a sound scientific basis for the better understanding of themechanism of shrimp disease resistance ability to WSSV infection, as well as thedevelopment of countermeasures for更多还原