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双孢蘑菇2796凝集素基因的扩增与原核表达

林勇 肖冬来 刘俊锋 唐小峦

林勇,肖冬来,刘俊锋,等. 双孢蘑菇2796凝集素基因的扩增与原核表达 [J]. 福建农业学报,2020,35(4):437−442 doi: 10.19303/j.issn.1008-0384.2020.04.011
引用本文: 林勇,肖冬来,刘俊锋,等. 双孢蘑菇2796凝集素基因的扩增与原核表达 [J]. 福建农业学报,2020,35(4):437−442 doi: 10.19303/j.issn.1008-0384.2020.04.011
LIN Y, XIAO D L, LIU J F, et al. Amplification and Expression of a Lectin Gene from Agaricus bisporus AS2796 [J]. Fujian Journal of Agricultural Sciences,2020,35(4):437−442 doi: 10.19303/j.issn.1008-0384.2020.04.011
Citation: LIN Y, XIAO D L, LIU J F, et al. Amplification and Expression of a Lectin Gene from Agaricus bisporus AS2796 [J]. Fujian Journal of Agricultural Sciences,2020,35(4):437−442 doi: 10.19303/j.issn.1008-0384.2020.04.011

双孢蘑菇2796凝集素基因的扩增与原核表达

doi: 10.19303/j.issn.1008-0384.2020.04.011
基金项目: 福建卫生职业技术学院应用技术协同创新研究重点项目(2019-5-2);福建省科技特派员项目(闽财指[2019]476号);福建省科技计划公益类专项(2015R1020-4)
详细信息
    作者简介:

    林勇(1970−),男,副研究员,研究方向:天然产物研究与应用(E-mail:lforesty@163.com

  • 中图分类号: S 646

Amplification and Expression of a Lectin Gene from Agaricus bisporus AS2796

  • 摘要:   目的  获得双孢蘑菇凝集素基因,了解双孢蘑菇凝集素家族蛋白的功能。  方法  根据已报道的双孢蘑菇凝集素基因序列设计引物,以总DNA为模板扩增双孢蘑菇凝集素基因。将长432 bp的基因亚型克隆后连接至携带His标签的原核表达载体pET-28a,转化大肠杆菌BL21(DE3),经IPTG诱导表达,表达产物经亲和层析纯化后,进行Western blot印迹分析。  结果  扩增的凝集素基因大小为432 bp,重组表达质粒pET-Lectin构建正确,重组凝集素蛋白在大肠杆菌BL21(DE3)中的表达产物约为18 kDa,重组蛋白纯化后纯度达95%以上。  结论  本研究首次扩增双孢蘑菇凝集素基因,并获得纯度较高的双孢蘑菇凝集素蛋白,可利用凝集试验检测其生物学活性,为目的基因的深入研究提供参考依据。
  • 图  1  琼脂糖凝胶电泳检测凝集素基因PCR产物

    注:M:DNA 分子量标准;1:PCR产物。

    Figure  1.  Detecting PCR products of lectin gene with agarose gel electrophoresis

    Note: M:DNA marker; 1:PCR product.

    图  2  重组质粒pMD-lectin双酶切鉴定

    注:M:DNA分子量标准;1~4:Bam HI和Xho Ⅰ双酶切重组子pMD-lectin。

    Figure  2.  Restriction enzyme digestion of pMD-lectin

    Note: M: DNA Marker, 1–4: restriction enzyme digestion of pMD-lectin by Bam HI and Xho Ⅰ.

    图  3  重组质粒pET-lectin的PCR验证

    注:M:DNA分子量标准;1~3:质粒PCR验证重组子pET-lectin。

    Figure  3.  PCR verification of pET-lectin

    Note: M: DNA Marker; 1–3: Plasmid PCR validation of pET-lectin.

    图  4  凝集素融合基因表达产物的SDS-聚丙烯酰胺电泳分析

    注:M:低分子量标准蛋白;1:含诱导空载体pET-28a的菌体总蛋白;2:含未诱导空载体pET-28α的菌体总蛋白;3~4:含未诱导重组载体pET-lectin的菌体总蛋白;5~8:含诱导重组载体pET-lectin的菌体总蛋白。

    Figure  4.  SDS-PAGE on expression product of lectin fusion gene

    Note: M: Protein Marker; 1: expression products of induced pET-28α; 2: expression products of pET-28α without induction; 3 and 4: expression products of recombinant pET-lectin without induction; 5–8: expression products of induced recombinant carrier pET-lectin.

    图  5  凝集素融合蛋白Western blot分析

    注:1:含未诱导重组载体pET-lectin的菌体总蛋白;2~4:含诱导重组载体pET-lectin的菌体总蛋白。

    Figure  5.  Western blot on lectin fusion protein

    Note: 1: total bacterial protein of recombinant carrier pET-lectin without induction; 2–4: total bacterial protein of induced recombinant carrier pET-lectin.

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