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中文摘要：
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c DNA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 k D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p ET-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m RNA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。
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