细菌基因组DNA对中华绒螯蟹（Eriocheir sinensis）免疫功能调节作用的研究
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摘要
大量研究表明,含未甲基化CpG基序的细菌基因组DNA和人工合成的CpG ODN是人和多种动物有效的免疫激活剂。但是,迄今尚未见有关细菌基因组DNA对中华绒螯蟹免疫功能影响的研究报道。
为探讨细菌基因组DNA对中华绒螯蟹蟹免疫功能及抗病力的影响,选择富含CpG序列的对中华绒螯蟹具有益生作用的枯草芽孢杆菌和具有致病作用的嗜水气单胞菌的基因组DNA进行试验。将两种细菌的基因组DNA分别按5、20、80μg/kg体重的剂量体腔注射中华绒螯蟹,以注射0.1 mL TE缓冲液的中华绒螯蟹为对照,在注射后第1、3、5、7 d采样,进行一系列免疫学指标的测定;并在第二次采样(第3 d)时,从每个处理组中取蟹9只,以1.2×107 cfu/kg体重的剂量体腔注射致病性嗜水气单胞菌CL99920菌株,记录接种7 d后各组蟹的累积死亡率。结果显示:注射一定剂量的两种细菌的基因组DNA均能显著地增加中华绒螯蟹的血细胞总数(THC),显著地增强血细胞呼吸爆发(O2-产量)和胞内酚氧化酶(POs、POT)活性,显著地增强血清酚氧化酶(PO)、超氧化物岐化酶(SOD)、酸性磷酸酶(ACP)、溶菌酶(LSZ)、一氧化氮合酶(NOS)活性,也能显著增强肝胰腺SOD活性和肌肉中的Na+-K+-ATP酶活性(P<0.05),但对血清碱性磷酸酶(ALP)活性没有显著影响(P>0.05)。两种细菌组DNA能不同程度地提高中华绒螯蟹对致病性嗜水气单胞菌的抗感染能力。在本试验条件下,两种细菌的基因组DNA均以20μg/kg剂量对中华绒螯蟹免疫力和抗病力的刺激效果最好,枯草芽孢杆菌基因组DNA和嗜水气单胞菌基因组DNA的免疫保护率分别为100%和71.43%。
Many studies indicate that unmethylated CpG motifs of bacterial genomic DNA and synthetic CpG ODN were a powerful activator of human and animal immune. However, the bacterial genomic DNA on the immune function of Chinese mitten crab reports was not yet seen to date.
In order to determine the immunomodulatory effects of bacterial genomic DNA in the Chinese mitten crab (Eriocheir sinensis), the experiment one was processed. Crab were coelom injected with 5, 20, 80μg genomic DNA of Bacillus substilis and genomic DNA of Aeromonas hydrophila kg?1 crab weight. A series of immunology parameters were examined at 1, 3, 5, and 7 days after administration of bacterial genomic DNA. At the same time, one group injected with 0.1mLTE was set as control. Crab were challenged at 1.2×107 colony forming units (cfu)/kg crab weight with a virulent strain of Aeromonas hydrophila (CL99920) of E. sinensis at the sencond sampling time (3d), and mortalities were recorded over a 7-day period. The results demonstrated that the groups injected with genomic DNA of Bacillus substilis and genomic DNA of Aeromonas hydrophila showed a remarkble higher number of THC than controls; while the respiratory burst (release of superoxide anion) increased significantly; also the serum parameters, including phenoloxidase (PO), superoxide dismutase (SOD), acid phosphatase (ACP), lysozyme (LSZ), Nitric oxide synthase (NOS), SOD of Hepatopancreas and Na+-K+-ATPase of muscle were assayed to be significantly increased than the controls(P<0.05). But alkaline phosphatase (ALP) was not significantly changed(P>0.05). At the same time, two kinds of bacteria genomic DNA treated crab’s ability of anti-infected by Aeromonas hydrophila were remarkably enhanced. Under this experimental condition, dose of 20μg / kg of two bacterial genomic DNA were best stimuluded in immunity and disease resistance of the Chinese mitten crab, while immune protection rate of the DNA of Bacillus substilis and genomic DNA of Aeromonas hydrophila on Chinese mitten crab was 100% and 71.43% respectively. The results showed that two bacterial genome DNAs were a new potential for mitten crab immunopotentiator. They were worth further developing and utilizing.
为探讨细菌基因组DNA对中华绒螯蟹蟹免疫功能及抗病力的影响,选择富含CpG序列的对中华绒螯蟹具有益生作用的枯草芽孢杆菌和具有致病作用的嗜水气单胞菌的基因组DNA进行试验。将两种细菌的基因组DNA分别按5、20、80μg/kg体重的剂量体腔注射中华绒螯蟹,以注射0.1 mL TE缓冲液的中华绒螯蟹为对照,在注射后第1、3、5、7 d采样,进行一系列免疫学指标的测定;并在第二次采样(第3 d)时,从每个处理组中取蟹9只,以1.2×107 cfu/kg体重的剂量体腔注射致病性嗜水气单胞菌CL99920菌株,记录接种7 d后各组蟹的累积死亡率。结果显示:注射一定剂量的两种细菌的基因组DNA均能显著地增加中华绒螯蟹的血细胞总数(THC),显著地增强血细胞呼吸爆发(O2-产量)和胞内酚氧化酶(POs、POT)活性,显著地增强血清酚氧化酶(PO)、超氧化物岐化酶(SOD)、酸性磷酸酶(ACP)、溶菌酶(LSZ)、一氧化氮合酶(NOS)活性,也能显著增强肝胰腺SOD活性和肌肉中的Na+-K+-ATP酶活性(P<0.05),但对血清碱性磷酸酶(ALP)活性没有显著影响(P>0.05)。两种细菌组DNA能不同程度地提高中华绒螯蟹对致病性嗜水气单胞菌的抗感染能力。在本试验条件下,两种细菌的基因组DNA均以20μg/kg剂量对中华绒螯蟹免疫力和抗病力的刺激效果最好,枯草芽孢杆菌基因组DNA和嗜水气单胞菌基因组DNA的免疫保护率分别为100%和71.43%。
Many studies indicate that unmethylated CpG motifs of bacterial genomic DNA and synthetic CpG ODN were a powerful activator of human and animal immune. However, the bacterial genomic DNA on the immune function of Chinese mitten crab reports was not yet seen to date.
In order to determine the immunomodulatory effects of bacterial genomic DNA in the Chinese mitten crab (Eriocheir sinensis), the experiment one was processed. Crab were coelom injected with 5, 20, 80μg genomic DNA of Bacillus substilis and genomic DNA of Aeromonas hydrophila kg?1 crab weight. A series of immunology parameters were examined at 1, 3, 5, and 7 days after administration of bacterial genomic DNA. At the same time, one group injected with 0.1mLTE was set as control. Crab were challenged at 1.2×107 colony forming units (cfu)/kg crab weight with a virulent strain of Aeromonas hydrophila (CL99920) of E. sinensis at the sencond sampling time (3d), and mortalities were recorded over a 7-day period. The results demonstrated that the groups injected with genomic DNA of Bacillus substilis and genomic DNA of Aeromonas hydrophila showed a remarkble higher number of THC than controls; while the respiratory burst (release of superoxide anion) increased significantly; also the serum parameters, including phenoloxidase (PO), superoxide dismutase (SOD), acid phosphatase (ACP), lysozyme (LSZ), Nitric oxide synthase (NOS), SOD of Hepatopancreas and Na+-K+-ATPase of muscle were assayed to be significantly increased than the controls(P<0.05). But alkaline phosphatase (ALP) was not significantly changed(P>0.05). At the same time, two kinds of bacteria genomic DNA treated crab’s ability of anti-infected by Aeromonas hydrophila were remarkably enhanced. Under this experimental condition, dose of 20μg / kg of two bacterial genomic DNA were best stimuluded in immunity and disease resistance of the Chinese mitten crab, while immune protection rate of the DNA of Bacillus substilis and genomic DNA of Aeromonas hydrophila on Chinese mitten crab was 100% and 71.43% respectively. The results showed that two bacterial genome DNAs were a new potential for mitten crab immunopotentiator. They were worth further developing and utilizing.
引文
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