凡纳滨对虾C-型凝集素基因原核表达与免疫作用的研究
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中国海洋大学 海水养殖教育部重点实验室,中国海洋大学 海水养殖教育部重点实验室,中国海洋大学 海水养殖教育部重点实验室

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教育部新世纪优秀人才支持计划(NCET-06-0597)


Prokaryotic expression of C-type lectin gene of Litopenaeus vannamei and immunologic activity evaluation of recombinant protein
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The Key Laboratory of Mariculture,Ministry of Education,Ocean University of China,The Key Laboratory of Mariculture,Ministry of Education,Ocean University of China,The Key Laboratory of Mariculture,Ministry of Education,Ocean University of China

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University of Ministry of Education of China(NCET-06-0597)

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    摘要:

    本文利用原核表达系统对凡纳滨对虾C-型凝集素基因的开放阅读框进行了重组表达,并通过纯化分离获得重组目的蛋白,采用血细胞悬浮培养的方法,研究了重组C-型凝集素对凡纳滨对虾血细胞免疫作用的影响。实验梯度设置为:对照组和重组C-型凝集素蛋白(1.0 mg/mL),分别在0、3、6、9、12、24h取培养的血细胞和培养液进行免疫相关指标的测定。结果表明:重组C-型凝集素经SDS-PAGE分析显示在39KD处有显著诱导条带,与预测的分子量大小基本一致,Western blot分析可以与anti-His抗体特异性结合,从而证明C-型凝集素重组蛋白在大肠杆菌中成功表达。重组C-型凝集素对大肠杆菌具有明显的凝集作用,对凡纳滨对虾血细胞数量、存活率和吞噬率影响显著(P<0.05);培养基酚氧化酶活力在培养3-12h内呈峰值变化,于9h时达到最大值(P<0.05)。研究表明,C-型凝集素不仅具有凝集作用,而且还能激活酚氧化酶原系统,具有重要的免疫防御作用。

    Abstract:

    The studies on innate immune responses of Litopenaeus vannamei C-type lectin, were gained by recombinant C-type lectin protein through expressed in Escherichia coli. We used cell suspension culture to study the effect of C-type lectin on the cellular immune response of Litopenaeus vannamei. The experiment was divided into two groups control group and recombinant protein (1.0 mg/mL). After 0, 3, 6, 9, 12, 24 hours, we harvested the solutions and then assessed parameters respectively. The results showed that SDS-PAGE revealed the molecular weight of LvLec protein was 39KD, which was consistent with the predicted size. Western blot revealed it could bind to anti-His antibody, which proved that C-lectin recombinant protein in Escherichia coli succeeded in expression. For the hemagglutination assay, massive agglutination could be observed after Escherichia coli was incubated with LvLec for 1 h. At the same time, Lvlec protein had a significant effect on haemocyte count, cell viability and phagocytic activity of white shrimp Litopenaeus vannamei(P<0.05). The extracelluar phenoloxidase activity of the Lvlec protein group showed peak changes during 3-12h, at 9h the extracelluar phenoloxidase activity was highest, then recovered to the control level(P<0.05). Therefore, crustacean C-type lectin not only had the function of hemagglutination , but also activated prophenoloxidase activating system, and probably played an important role in immune response.

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于金红,潘鲁青,张辉.凡纳滨对虾C-型凝集素基因原核表达与免疫作用的研究[J].水产学报,2013,37(8):1183~1191

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  • 收稿日期:2013-01-31
  • 最后修改日期:2013-04-07
  • 录用日期:2013-05-07
  • 在线发布日期: 2013-08-30
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