对虾血细胞中一氧化氮合成酶鉴定与分析方法研究

Studies on the methods of identification and activity assay of inducible nitric oxide synthase in haemocytes of shrimp

通过硝基蓝四唑(NBT)还原法和血细胞形态法两种一氧化氮合成酶的鉴定方法,以日本对虾(Penaeusjaponicus)作为研究对象,对日本对虾血细胞中的一氧化氮合成酶进行初步鉴定,并优化硝基蓝四唑(NBT)还原法测定对虾血细胞中一氧化氮合成酶的实验条件。结果显示,当L-精氨酸浓度为2.5mmol/L,脂多糖(LPS)质量浓度为100μg/mL,Ca2+浓度为2.5mmol/L时,NBT法测定一氧化氮合成酶活力的结果最佳。同时,还建立了两种针对一氧化氮合成酶活力的分析方法—L-瓜氨酸分析法和亚硝酸盐分析法,并对其测定条件进行了优化。结果表明,对虾血细胞在4℃下与L-精氨酸和LPS孵育8h后测定的结果最为理想。利用这4种方法能够有效地鉴定和分析对虾血细胞中的一氧化氮合成酶,这为深入了解一氧化氮合成酶在对虾先天性免疫中的地位及其在对虾抗病力中的作用提供了理论依据。

The study of shrimp immunology in response to pathogen infection is necessary for the sound management strategies in controlling shrimp diseases and elevating the production. Shrimp is lack of adaptive immune system, and the disease resistance ability relies mainly on the non-specific innate immune system, among which NOS system should play a very important role. NOS system could produce reactive nitrogen intermediates (RNIs) which have the anitiviral, antibacterial and anti-parasite properties. However, the role of NOS in shrimp immunological system is seldom reported. In our present studies, four methods previously reported in identification and assay of inducible NOS (iNOS) in other creatures have been reestablished and modified for the application in shrimps. The two methods based on NBT-reduction reaction and cellular morphological observation were employed to identify the iNOS in haemocytes of Penaeus japonicus according to Weiske et al (1999) and Franchini et al (1995). Both of the methods showed that shrimp haemocytes have the activity of iNOS, especially after induction with LPS. The conditions for NBT method were modified according to the experimental results, and the optimal concentrations of L-arginine, LPS and Ca^(2+) were 2.5 mmol/L, 100 μg/mL and 2.5 mmol/L, respectively. The results of cellular morphological observation showed that haemocytes treated with LPS had the similar effects with that treated with NO donor—SNP, which suggested the existence of iNOS in the haemocytes of P. japonicus. The effect could be blocked with the addition of NOS inhibitor further confirmed the result.The two methods based on the analysis of L-citrulline and NO_2^- were used in the experiment for assay of iNOS activity in haemocytes of shrimps quantitatively. The standard curve for measurement of L-citrulline and NO_2^- were established for the analysis, and the incubation time for the assay of iNOS enzyme activity was optimized. The results showed that 8 h incubation with arginine and LPS was necessary, and enzyme activity of iNOS would not be well induced if the incubation time was not enough. The two methods could get comparable results under controlled experimental conditions. Therefore, either of the method could be used in the following experiment to assay the variation of iNOS enzyme activity under different conditions.The reestablished methods have identified the iNOS activity in haemocytes of P. japonicus and analyzed the enzyme activity effectively. The results of the expeirment would provide a good way for the further studies of iNOS in immunology and disease resistance ability of shrimps.