文章摘要
崔峥,朱小玲,邱高峰.日本沼虾VASA蛋白的原核表达、抗体制备及其免疫鉴定[J].水产学报,2010,34(10):1495~1501
日本沼虾VASA蛋白的原核表达、抗体制备及其免疫鉴定
Prokaryotic expression,antibody preparation and immunological characterization of VASA protein in the prawn Macrobrachium nipponense
投稿时间:2010-03-31  修订日期:2010-06-18
DOI:10.3724/SP.J.1231.2010.06894
中文关键词: 日本沼虾  VASA蛋白  原核表达  多克隆抗体  免疫鉴定
英文关键词: Macrobrachium nipponense  VASA protein  prokaryotic expression  polyclonal antibody  immunological characterization
基金项目:上海市浦江人才计划项目(08PJ1409000);上海市科委自然科学基金项目(07JR14102);上海市教委重点学科建设项目(J50701);上海市教委科研创新项目(08ZZ80)
作者单位E-mail
崔峥 上海海洋大学农业部水产种质资源与利用重点开放实验室  
朱小玲 上海海洋大学农业部水产种质资源与利用重点开放实验室  
邱高峰 上海海洋大学 gfqiu@shou.edu.cn 
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中文摘要:
      Vasa基因具有在性腺中特异表达的特点,在生殖细胞分化与发育过程中起重要作用。从日本沼虾精巢cDNA中克隆了vasa基因的阅读框,连接到pET-32a载体,构建重组表达质粒pET32a-vasa,转化大肠杆菌BL21,经IPTG诱导融合表达,表达产物经SDS-PAGE分析表明,融合蛋白主要以包涵体形式存在,分子量约85 ku,表达量约占包涵体总蛋白的48.7%,Western blotting检测表明,融合蛋白可特异地被anti-HIS标签抗体识别。用Ni2+-NTA纯化后的融合蛋白免疫家兔,制备获得多克隆抗体,ELISA显示该抗体效价达1∶160 000,Western免疫鉴定显示该抗体不仅能识别融合蛋白,而且也能识别日本沼虾性腺粗提液中的内源性VASA蛋白,免疫组化进一步显示,VASA蛋白主要分布在卵母细胞核周围和精原细胞质中,成熟精子中无信号,暗示VASA在日本沼虾生殖细胞发育分化过程中扮演重要角色。
英文摘要:
      Vasa gene is specifically expressed in gonads and plays an important role in the germline formation and differentiation in most animals.In this study,a full open reading frame of vasa gene was amplified from the testis cDNA pool of the prawn Macrobrachium nipponense,and ligated with expression plasmid pET32a with 6×histine tag.After the recombinant plasmid was transferred into host bacteria Escherichia coli (BL21),the recombinant VASA protein was expressed under the induction of IPTG.The optimum concentration of IPTG was 0.2 mmol/L with a duration of 6 h at 30 ℃.SDS PAGE analysis showed that the fusion protein was highly expressed as inclusion body and the yield accounted for 48.7% of the total bacterial protein.Western blotting analysis demonstrated the target protein with the molecular weight of 85 ku.The recombinant protein was subsequently purified by Ni2+-NTA affinity chromatography.To further prepare the prawn VASA antibody,New Zealand white rabbits were immuned by the purified protein.The highest titer of the antiserums was up to1∶160 000as revealed by ELISA assay.The specificity of the antibodies against VASA protein was verified by Western blotting.Immunohistochemical analyses indicated that,in oocyte VASA protein was mainly concentrated at the perinuclear region at vitellogenic oocyte; in testis,VASA protein was mainly distributed in spermatogonia.These data suggest that the VASA could play a key role in the development and differentiation of the germ cells in the prawn.
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