抗肌肉乳化病候选品系三疣梭子蟹几个免疫因子的研究
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摘要
三疣梭蟹营养丰富,是重要的经济水产动物,但是肌肉乳化病严重影响了梭子蟹的养殖效益。为此我实验室用肌肉乳化病的致病菌-溶藻弧菌对其连续筛选育种2代(以下简称筛选蟹F1, F2),家系选育第3代(以下简称筛选蟹F3)。本文以抗脂多糖因子(ALF),血蓝蛋白,血清中的重要免疫酶类,如水解酶:酸性磷酸酶(ACP)、碱性磷酸酶(AKP),保护酶:超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和酚氧化酶(PO)为指标,采用半定量RT-PCR方法测定ALF表达量,用紫外分光光度法测定血蓝蛋白含量,用试剂盒测定各血清免疫酶活力的方法,研究了3代筛选蟹的免疫防御机制。同期的普通梭子蟹作为对照。结果如下:
1.溶藻弧菌感染6h对筛选蟹F1代肝胰脏血清中免疫酶活力和ALF表达量的影响。
筛选蟹F1代和同期的普通蟹血清中免疫酶活性在12-36h达到最大值;肝胰脏中ALF表达量分别在从感染开始计9h和12h达到最大值,然后逐渐下降;各免疫因子可被溶藻弧菌激活,并且存在明显的时间依赖性。筛选蟹F1的血清免疫酶活力和ALF表达量均优于同期的普通蟹。
2.温度和盐度胁迫对筛选蟹F2代血蓝蛋白和血清中免疫酶活力的影响
温度和盐度胁迫下,筛选蟹F2代和同期的普通蟹应激反应强烈,血蓝蛋白含量和血清免疫酶活力随时间表现出有升有降的不同变化趋势。温度和盐度胁迫对三疣梭子蟹非特异性免疫影响显著,筛选蟹F2代的血蓝蛋白含量和血清免疫酶活力均显著高于同期的普通蟹,筛选蟹F2比普通蟹F1在非特异性免疫因子方面具明显的免疫优势。
3.筛选蟹F3代养殖期不同发育阶段ALF表达量研究
筛选蟹F3代和同期的普通蟹ALF在溞状幼体阶段（1-4)变化较小;从溞
4-大眼幼体阶段,ALF表达量显著上升(P>0.05);大眼幼体—稚蟹阶段无明显变化,但此时ALF表达量为所有发育阶段中最大。三疣梭子蟹从溞状幼体开始的整个发育阶段均有ALF表达,证明其为组成性表达,是免疫体系中一个非常重要且必需的组成成分,在其各个发育阶段扮演着帮助机体抵御外来微生物入侵的角色。在溞状幼体-稚蟹整个发育阶段,筛选蟹F3和同期普通蟹的ALF表达量没有显著差异。但是从大眼幼体开始,筛选蟹F3的ALF表达量高于普通蟹F1,可能从大眼幼体开始逐渐显现免疫优势。
Portunus trituberculatus, nutrient-rich , is one of important economic aquatic animals. Emulsification disease of Portunus trituberculatus has seriously effected its aquaculture efficiency. So our laboratory screened Portunus trituberculatus by Vibrio alginolyticus, the pathogens of emulsification disease; and breed F1, F2( short as PSVF1, PSVF2 as followings). F3 ( short as PSVF3 as followings) was pedigree breeded the next year. This paper studied the immune defense mechanism of F1, F2 and F3 focusing on the immune factors such as anti-lipopolysaccharide factor (ALF); hemocyanin; isoenzyme, like acid phosphatase (ACP), alkaline phosphatase (AKP); protective enzymes, like superoxide dismutase (SOD), peroxidase (POD), catalase(CAT) and phenoloxidase (PO). Semi-quantitive RT-PCR was used to analyze the ALF expression; UV Spectrophotometry and enzyme analysis method was respectively used to study the hemocyanin content and the serum enzymes activities. Commom Portunus trituberculatus in the same period was served as control(short as CPF1 as followings). The main results are as followings: 1.The effect of infection 6h by Vibrio alginolyticus on ALF expression in hepatopancreas, serum immunoenzymes activities of PSVF1.
ALF expressions in hepatopancreas of PSVF1 and CPF1 reached maximun respectivly at 9h and 12h counting the infection time of 6h, and then gradually decline; serum immunoenzymes activities of PSVF1 and CPF1 reached maximum at 12-36h; The time dependency after infection was obvious. PSVF1 has more immunity advantage on the hemocyanin content and serum immunoenzymes than that of CPF1. 2. Effects of temperature and salinity stress on the content of hemocyanin and serum immunoenzymes activities of PSVF2.
The result showed that under temperature and salinity stress, PSVF2 and CPF1 showed strong stress response, and the non-specific immunity was significantly affected; The content of hemocyanin and serum immunoenzymes activities of PSVF2 were higher than that of CPF1, and PSVF2 showed more non-specific immunity advantages. 3.Expressions of ALF during the growth period of PSVF3
ALF expressions throughout the four zoea stages had no significant difference, then rapidly up-regulated at megalops stage (P>0.05), reached maxium at the first and second juvenile crab stages of both PSVF3 and CPF1. ALF transcripts were present in all detected samples during different developmental stages. This suggests that ALF is constitutively expressed in Portunus trituberculatus, it may also play an important role in innate defense against infection. During the whole growth period from zoea to junivile crab, there were no distinctively difference in ALF expressions between PSVF3 and CPF1. However, ALF expressions of megalops and juvenile cab stages of PSVF3 were higher than that of CPF1, this may suggest that PSVF3 showed more immunity advantage beginnig from megalops stages.
1.溶藻弧菌感染6h对筛选蟹F1代肝胰脏血清中免疫酶活力和ALF表达量的影响。
筛选蟹F1代和同期的普通蟹血清中免疫酶活性在12-36h达到最大值;肝胰脏中ALF表达量分别在从感染开始计9h和12h达到最大值,然后逐渐下降;各免疫因子可被溶藻弧菌激活,并且存在明显的时间依赖性。筛选蟹F1的血清免疫酶活力和ALF表达量均优于同期的普通蟹。
2.温度和盐度胁迫对筛选蟹F2代血蓝蛋白和血清中免疫酶活力的影响
温度和盐度胁迫下,筛选蟹F2代和同期的普通蟹应激反应强烈,血蓝蛋白含量和血清免疫酶活力随时间表现出有升有降的不同变化趋势。温度和盐度胁迫对三疣梭子蟹非特异性免疫影响显著,筛选蟹F2代的血蓝蛋白含量和血清免疫酶活力均显著高于同期的普通蟹,筛选蟹F2比普通蟹F1在非特异性免疫因子方面具明显的免疫优势。
3.筛选蟹F3代养殖期不同发育阶段ALF表达量研究
筛选蟹F3代和同期的普通蟹ALF在溞状幼体阶段（1-4)变化较小;从溞
4-大眼幼体阶段,ALF表达量显著上升(P>0.05);大眼幼体—稚蟹阶段无明显变化,但此时ALF表达量为所有发育阶段中最大。三疣梭子蟹从溞状幼体开始的整个发育阶段均有ALF表达,证明其为组成性表达,是免疫体系中一个非常重要且必需的组成成分,在其各个发育阶段扮演着帮助机体抵御外来微生物入侵的角色。在溞状幼体-稚蟹整个发育阶段,筛选蟹F3和同期普通蟹的ALF表达量没有显著差异。但是从大眼幼体开始,筛选蟹F3的ALF表达量高于普通蟹F1,可能从大眼幼体开始逐渐显现免疫优势。
Portunus trituberculatus, nutrient-rich , is one of important economic aquatic animals. Emulsification disease of Portunus trituberculatus has seriously effected its aquaculture efficiency. So our laboratory screened Portunus trituberculatus by Vibrio alginolyticus, the pathogens of emulsification disease; and breed F1, F2( short as PSVF1, PSVF2 as followings). F3 ( short as PSVF3 as followings) was pedigree breeded the next year. This paper studied the immune defense mechanism of F1, F2 and F3 focusing on the immune factors such as anti-lipopolysaccharide factor (ALF); hemocyanin; isoenzyme, like acid phosphatase (ACP), alkaline phosphatase (AKP); protective enzymes, like superoxide dismutase (SOD), peroxidase (POD), catalase(CAT) and phenoloxidase (PO). Semi-quantitive RT-PCR was used to analyze the ALF expression; UV Spectrophotometry and enzyme analysis method was respectively used to study the hemocyanin content and the serum enzymes activities. Commom Portunus trituberculatus in the same period was served as control(short as CPF1 as followings). The main results are as followings: 1.The effect of infection 6h by Vibrio alginolyticus on ALF expression in hepatopancreas, serum immunoenzymes activities of PSVF1.
ALF expressions in hepatopancreas of PSVF1 and CPF1 reached maximun respectivly at 9h and 12h counting the infection time of 6h, and then gradually decline; serum immunoenzymes activities of PSVF1 and CPF1 reached maximum at 12-36h; The time dependency after infection was obvious. PSVF1 has more immunity advantage on the hemocyanin content and serum immunoenzymes than that of CPF1. 2. Effects of temperature and salinity stress on the content of hemocyanin and serum immunoenzymes activities of PSVF2.
The result showed that under temperature and salinity stress, PSVF2 and CPF1 showed strong stress response, and the non-specific immunity was significantly affected; The content of hemocyanin and serum immunoenzymes activities of PSVF2 were higher than that of CPF1, and PSVF2 showed more non-specific immunity advantages. 3.Expressions of ALF during the growth period of PSVF3
ALF expressions throughout the four zoea stages had no significant difference, then rapidly up-regulated at megalops stage (P>0.05), reached maxium at the first and second juvenile crab stages of both PSVF3 and CPF1. ALF transcripts were present in all detected samples during different developmental stages. This suggests that ALF is constitutively expressed in Portunus trituberculatus, it may also play an important role in innate defense against infection. During the whole growth period from zoea to junivile crab, there were no distinctively difference in ALF expressions between PSVF3 and CPF1. However, ALF expressions of megalops and juvenile cab stages of PSVF3 were higher than that of CPF1, this may suggest that PSVF3 showed more immunity advantage beginnig from megalops stages.
引文
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