| 摘要: |
| 诱导型一氧化氮合成酶(iNOS)在生物机体免疫，特别在无脊椎动物免疫中的作用近来得到了广泛的关注，由其催化产生的一氧化氯(NO)除具有已知的神经传导、松弛平滑肌等功能外，还具有抗菌、抗病毒、抗寄生虫等作用。作者通过硝基四氯唑蓝(NBT)法和血细胞形态观察等方法，对中国明对虾(Fenneropenaeus chinensis)血细胞中存在的诱导型一氧化氮合成酶进行了初步鉴定。在此基础上，通过亚硝酸盐法和L-瓜氨酸法对比，研究了感染白斑综合症病毒(WSSV)后中国明对虾血细胞中一氧化氯合成酶的变化情况。结果显示，中国明对虾在感染WSSV后，iNOS活性在12h内有上升趋势，实验36h后酶活性显著下降，至60h后酶活性降至对照组的一半左右。同时，被脂多糖(LPS)诱导的一氧化氮合成酶活性与对照相比也有显著下降。与此对应的是，核酸探针斑点杂交法检测病毒的结果显示：实验36h后在对虾体内能够检测到白斑综合症病毒。对照组中国明对虾血细胞的iNOS在实验过程中基本保持稳定。这说明WSSV在感染中国明对虾初期可以诱导血细胞产生iNOS，但随着WSSV在中国明对虾体内的大量增殖及其对血细胞的破坏，使得iNOS活性显著降低，对虾也趋于死亡。因此，iNOS能够作为反映对虾在病毒感染过程中健康状况的有效指标。 |
| 关键词: 中国明对虾,一氧化氮合成酶,自斑综合症病毒 |
| DOI： |
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| 基金项目:国家重点基础研究项目资助，G1999012011号 |
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| IDENTIFICATION OF INDUCIBLE NITRIC OXIDE SYNTHASE(iNOS) IN HAEMOCYTES OF SHRIM P FENNEROPENAEUS CHINENSIS AND ENZYME ACTIVITY DURING ITS INFECTED BY WHITE SPOT SYNDROME VIRUS |
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JIANG Guo-Jian1,2, YU Ren-Cheng1, WANG Yun-Feng1, YAN Tian1, ZHOU Ming-Jiang1
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1.Key Laboratory of Marine Ecology and Environmental Sciences，Institute of Oceanology，Chinese Academy of Sciences;2.Graduate School，Chinese Academy of Sciences
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| Abstract: |
| In recent years, much attention has been given to nitric oxide synthase(NOS)that catalyzes L-arginine to nitric oxide(NO). NOSs play significant roles in neurotransmission and smooth-muscle relaxation (constitutive NOS or cNOS) and in immunity(induced NOS or iNOS) in anti microbia, antivirus and anti-parasite. NOS has been considered as an important part in innate immune system, especially in that of invertebrates.
In this study, iNOS, induced by lipopolysaccharide(LPS), was detected in haemocytes of Fenneropenaeus chinensis, by NBT reduction and haemocyte morphological studies. NBT reduction began with fixation by 4% formaldehyde, and then incubated in NADPH, induced by LPS. The induced haemocytes of F. chinensis have an ability to reduce NBT at higher levels than that treated with no NADPH but NADH (the differential significance P<0. 001). Results of haemocyte morphological studies showed that haemocytes of F. chinensis mainly are in amoeboid conformation with sporadic Escherichia coli congregated or clumped in their immediate vicinity. Treating haemocytes with LPS stimulates bacterial clumping around the big and round haemocytes. But by adding NOS inhibitor L-NMMA, the bacterial clumping resulting from LPS was stopped and the haemocytes returned to amoeboid conformation again. Adding nitric oxide donor-SNP stimulated both bacterial clumping around the haemocytes and promoting and maintaining haemocytes in big and round configuration. These results concluded that LPS has the same effect on haemocytes of F. chinensis to NO donor-SNP. This effect can be hindered by NOS inhibitor. Therefore, the haemocytes of F. chinensis have LPS-inducible NOS.
Variation of iNOS enzyme activity was compared between healthy and WSSV (White Spot Syndrome Virus) infected shrimps using L-citrulline analysis and nitrite analysis. Results of both methods show that iNOS activity in haemocytes of F. chinensis had a general decreasing trend compared to the relatively stable value in healthy shrimps. iNOS activity in WSSV infected shrimp increased slightly 12 hours post-inoculation, but decreased sharply after 36 hours. Thereafter, decreasing gradually until the end of experiment when the shrimp died. Enzyme activity was only half that of the control 60 hours post-inoculation. In addition, NOS differences of LPS-induced with non-induced haemocytes in healthy F. chinensis are generally higher than that of the WSSV-infected shrimp. Meanwhile, LPS-induced iNOS activity also decreased significantly with passing time in WSSV infected shrimps. Detection of WSSV with the Dig-DNA Probe dot blot hybridization test showed W SSV could not be detected in F. chinensis until 36 hours after inoculation. These results suggest that WSSV infection can stimulate iNOS synthesis slightly in F. chinensis However, enzyme activity decreased sharply after proliferation of the virus, and the shrimp became both moribund and died simultaneously. iNOS activity of haemocytes of F. chinensis closely correlated with the infection and proliferation of WSSV. Therefore, iNOS can be considered as a valuable index for indicating health status and immune-competence of F. chinensis. |
| Key words: Fenneropenaeus chinensis, Induced nitric oxide synthase(iNOS), White Spot Syndrome Virus(WSSV) |
