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顾伟, 陈静, 黄艳青, 靳明建, 孟庆国, 王文. 红螯螯虾酚氧化酶原及其特性研究[J]. 水生生物学报, 2015, 39(1): 117-125. DOI: 10.7541/2015.15
引用本文: 顾伟, 陈静, 黄艳青, 靳明建, 孟庆国, 王文. 红螯螯虾酚氧化酶原及其特性研究[J]. 水生生物学报, 2015, 39(1): 117-125. DOI: 10.7541/2015.15
Gu Wei, Chen Jing, Huang Yan-qing, Jin Ming-jian, Meng Qing-guo, Wang Wen. The characterization of prophenoloxidase and phenoloxidase from red claw crayfish, Cherax quadricarinatus[J]. ACTA HYDROBIOLOGICA SINICA, 2015, 39(1): 117-125. DOI: 10.7541/2015.15
Citation: Gu Wei, Chen Jing, Huang Yan-qing, Jin Ming-jian, Meng Qing-guo, Wang Wen. The characterization of prophenoloxidase and phenoloxidase from red claw crayfish, Cherax quadricarinatus[J]. ACTA HYDROBIOLOGICA SINICA, 2015, 39(1): 117-125. DOI: 10.7541/2015.15

红螯螯虾酚氧化酶原及其特性研究

The characterization of prophenoloxidase and phenoloxidase from red claw crayfish, Cherax quadricarinatus

  • 摘要: 采用同源克隆策略和RACE技术, 从红螯螯虾Cherax quadricarinatus血细胞中克隆得到酚氧化酶原基因的全长cDNA序列, 共2951 bp, 开放读码框为1995 bp, 编码665个氨基酸. 预测的分子量和等电点分别为75.7 kD和6.23. 酚氧化酶原含有两个推测的tyrosinase copper-binding motifs (带有六个组氨酸残基)和一个thiol-ester-like motif, 这些特征和其他甲壳动物的酚氧化酶原特征相同. 红螯螯虾酚氧化酶原氨基酸序列与通讯螯虾Pacifastacus leniusculus、欧洲龙虾Homarus gammarus、美洲龙虾Homarus americanus 和克氏原螯虾Procambarus clarkii 酚氧化酶原的相似率分别为68%、63%、63%和59%. 酚氧化酶原基因双酶切后连接入pET-28a原核表达载体, 转化到大肠杆菌BL21后重组表达酚氧化酶原蛋白. 在重组蛋白纯化后, 免疫新西兰大耳兔制备得到的酚氧化酶原多克隆抗体, 其效价大于1:12800. 红螯螯虾血淋巴、肝和鳃组织中的酚氧化酶原mRNA表达和酚氧化酶活性较高, 而神经、心、肠和肌肉中较低. 中华绒螯蟹螺原体和嗜水气单胞菌免疫红螯螯虾后, 血淋巴细胞、肝和鳃组织中的酚氧化酶原和酚氧化酶活性在免疫后的不同时间均出现了显著性的增加, 此结果表明酚氧化酶原和酚氧化酶在红螯螯虾对抗细菌感染的过程中起到重要的免疫作用. 此结果为进一步深入研究酚氧化酶原基因和酚氧化酶的功能及其调控机理奠定基础.

     

    Abstract: In this study, a prophenoloxidase (proPO) gene was cloned from hemocytes of red claw crayfish, Cherax quadricarinatus. The open reading frame (ORF) of proPO gene is 1995 bp and encodes a 665 amino acids (aa) protein. The predicted molecular mass of the protein was 75.7 kD with an estimated pI of 6.23. It contained two putative tyrosinase copper-binding motifs with six histidine residues and one thiol-ester-like motif, which showed similar structural features of proPOs from other crustaceans. The amino acid sequence of C. quadricarinatus proPO showed similarity of 68%, 63%, 63% and 59% with that of the proPO of Pacifastacus leniusculus, Homarus gammarus, Homarus americanus and Procambarus clarkii, respectively. The recombinant proPO were expressed in Escherichia coli BL21 with pET-28a expression vector. After protein purification and antibody prepared, the titer of rabbit anti-proPO serum was above 1:12800. The mRNA transcription of proPO and PO activity in C. quadricarinatus were high in hemocytes, hepatopancreas and gills, lower values were seen in other tissues (nerve, heart, intestine and muscle). The immune characterization of proPO gene was studied after Spiroplasma eriocheiris and Aeromonas hydrophila stimulation. After S. eriocheiris or A. hydrophila challenging, the mRNA transcription of proPO gene and PO activity in hemocytes, hepatopancreas and gill of C. quadricarinatus were up-regulated at different times to resist different stimulation. These results indicated that it was potentially involved in the acute response against invading bacteria in C. quadricarinatus.

     

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