| 摘要: |
| 从cDNA 文库中获得了梅氏新贝尼登虫卵黄铁蛋白(NmYF)的编码序列。序列全长739 个核苷酸, 3′-末端具有polyA 尾, 单一大开放阅读框编码一个由226 个氨基酸组成的分子量为26.1kDa的蛋白。序列比较表明, NmYF 与卫氏并殖吸虫卵黄铁蛋白最相似, 氨基酸序列同源性为23.7%。系统进化树分析表明, NmYF、卫氏并殖吸虫卵黄铁蛋白和头槽绦虫卵黄铁蛋白形成了一个小的进化簇, 而体铁蛋白序列形成了一个大的进化簇, 揭示了卵黄铁蛋白和体铁蛋白间的差异以及进化上的相关性。这是梅氏新贝尼登虫卵黄铁蛋白基因序列的首次报道。随后, 构建了插入有全长NmYF 基因开放阅读框序列的原核表达质粒pET-22b-NmYF, 转化大肠杆菌BL21 pLys E 菌株, 经IPTG 诱导表达后, SDS-PAGE 检测表明预期大小的目的蛋白大量表达。随后制备了目的蛋白的多克隆抗血清,它能与目的蛋白起强的特异性反应, 但与细菌自身蛋白不起反应, 可用于后续研究。
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| 关键词: 梅氏新贝尼登虫, 卵黄铁蛋白, 序列分析, 原核表达, 抗血清制备 |
| DOI：10.11693/hyhz201001017017 |
| 分类号: |
| 基金项目:国家高技术研究与发展计划(863 计划)项目, 2006AA020701 号; 教育部“新世纪优秀人才支持计划”项目, NCET-08-0928号; 浙江省科技厅重点项目, 2008C22049 号 |
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| YOLK FERRITIN FROM NEOBENEDENIA MELLENI: cDNA CLONING, PROKARYOTIC EXPRESSION AND ANTISERUM PREPARATION |
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NIU He, CHEN Jiong, SHI Yu-Hong, LI Ming-Yun
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Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University
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| Abstract: |
| A full-length cDNA clone of yolk ferritin gene was isolated from the cDNA library of Neobenedenia melleni and tentatively named as NmYF, which was the first report of yolk ferritin gene from N. melleni. It was 739bp long and contained a large open reading frame (ORF) encoding a 26.1kDa protein. The NmYF was very similar to yolk ferritin of Paragonimus westermani in the sequence, and formed a small cluster of phylogenetic tree with yolk ferritins of P. westermani and Bothriocephalus acheilognathi, while soma ferritins formed another big cluster, showing distinct difference between yolk ferritin and soma ferritin. Furthermore, the prokaryotic expression plasmid pET-22b-NmYF containing the intact ORF was constructed and the aim protein was induced by IPTG. The SDS-PAGE analysis showed that the aim protein could be highly expressed in the expected size. A polyclonal antiserum raised to the aim protein reacted strongly with itself by western blot.
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| Key words: Neobenedenia melleni, Yolk ferritin, Sequence analysis, Prokaryotic expression, Antiserum preparation |
