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α6/α3β2β3乙酰胆碱受体在非洲爪蟾卵母细胞中的表达及其条件优化

施西子 毛楷林 朱晓鹏 长孙东亭 罗素兰

施西子, 毛楷林, 朱晓鹏, 长孙东亭, 罗素兰. α6/α3β2β3乙酰胆碱受体在非洲爪蟾卵母细胞中的表达及其条件优化[J]. 华南师范大学学报(自然科学版), 2020, 52(3): 70-77. doi: 10.6054/j.jscnun.2020046
引用本文: 施西子, 毛楷林, 朱晓鹏, 长孙东亭, 罗素兰. α6/α3β2β3乙酰胆碱受体在非洲爪蟾卵母细胞中的表达及其条件优化[J]. 华南师范大学学报(自然科学版), 2020, 52(3): 70-77. doi: 10.6054/j.jscnun.2020046
SHI Xizi, MAO Kailin, ZHU Xiaopeng, ZHANGSUN Dongting, LUO Sulan. The Expression of α6/α3β2β3 Acetylcholine Receptor in Xenopus laevis Oocytes and the Conditions for Its Optimization[J]. Journal of South China normal University (Natural Science Edition), 2020, 52(3): 70-77. doi: 10.6054/j.jscnun.2020046
Citation: SHI Xizi, MAO Kailin, ZHU Xiaopeng, ZHANGSUN Dongting, LUO Sulan. The Expression of α6/α3β2β3 Acetylcholine Receptor in Xenopus laevis Oocytes and the Conditions for Its Optimization[J]. Journal of South China normal University (Natural Science Edition), 2020, 52(3): 70-77. doi: 10.6054/j.jscnun.2020046

α6/α3β2β3乙酰胆碱受体在非洲爪蟾卵母细胞中的表达及其条件优化

doi: 10.6054/j.jscnun.2020046
基金项目: 

国家自然科学基金项目 81872794

国家自然科学基金项目 41966003

高等学校学科创新引智计划资助项目 D20010

详细信息
    通讯作者:

    罗素兰,教授,Email:luosulan2003@163.com

  • 中图分类号: Q812; Q786

The Expression of α6/α3β2β3 Acetylcholine Receptor in Xenopus laevis Oocytes and the Conditions for Its Optimization

  • 摘要: 为建立有效的α6/α3β2β3 nAChRs(α6/α3β2β3,或简写为α6β2*,*代表其他亚基)体外重组表达实验模型,利用非洲爪蟾卵母细胞表达体系,采用显微注射RNA的方法,利用双电极电压钳技术检测电流,对α6/α3β2β3 nAChRs亚型进行体外重组表达研究,并对其表达条件进行优化,获得了α6/α3β2β3 nAChRs优化的重组表达体系.烟碱型乙酰胆碱受体(nAChRs)是一类重要的配体门控离子通道,其亚型众多且结构相似,但各亚型的生理学和药理学功能截然不同.天然的α6β2* nAChRs很难体外表达,体外表达模型的表达电流非常小.因此, 本实验拟研究优化该亚型的表达,进行优化后的体外表达模型,产生的激发电流显著提高,为以该亚型为靶点的新药筛选提供实验模型和基础.
  • 图  1  含有nAChRs亚基基因的质粒及其酶切线性化产物的电泳图

    M: DNA Marker DL 10 000; 1: rα6/α3质粒; 2: rα6/α3质粒经SalⅠ酶切后; 3: rβ2质粒; 4: rβ2质粒经Afl Ⅲ酶切后; 5:rβ3质粒; 6:rβ3质粒经NheⅠ酶切后

    Figure  1.  The agarose gel electrophoresis of nAChRs plasmids and their linearized DNA

    图  2  nAChRs亚基rα6/α3、rβ2和rβ3的cRNA电泳图

    M:DNA Marker DL 15 000;1:rα6/α3 cRNA; 2:rβ2 cRNA; 3:rβ3 cRNA

    Figure  2.  The agarose gel electrophoresis of rα6/α3, rβ2 and rβ3 cRNA

    图  3  非洲爪蟾卵母细胞的酶解效果图

    Figure  3.  The Xenopus laevis oocytes after enzymolysis

    图  4  单亚基cRNA质量浓度、注射体积和ACh浓度对α6/α3β2β3受体电流表达的影响

    Figure  4.  The effect of cRNA concentration (single subunit), injection volume and ACh concentration on the current of α6 / α3β2β3 receptor

    图  5  α-芋螺毒素TxIB对α6/α3β2β3 nAChRs的阻断情况

    注:箭头所指为药物阻断后电流.

    Figure  5.  The blocking of α6/α3β2β3 nAChRs by α-CTX TxIB

    表  1  鼠源α6/α3、β2、β3 nAChRs亚基质粒对应的限制性内切酶

    Table  1.   The restriction endonuclease of plasmid which contained α6/α3, β2, β3 nAChRs subtypes(rat)

    包含的亚基 质粒载体 启动子 限制性内切酶
    rα6/α3 pT7TS T7 Sal
    rβ2 pGEMHE T7 Afl
    rβ3 pGEMHE T7 Nhe
      注:r为大鼠(rat)来源.
    下载: 导出CSV

    表  2  nAChRs亚基r α6/α3、r β2、r β3基因的质粒、线性化DNA和体外转录cRNA的质量浓度

    Table  2.   The concentration of nAChRs subunit α6/α3, β2 and β3 plasmid, linearized DNA and cRNA

    亚基基因 ρ(质粒DNA)/(μg·μL-1) ρ(线性化DNA)/(μg·μL-1) cRNA
    ρ/(μg·μL-1) A260/A280
    rα6/α3 0.38 0.22 0.88 2.18
    rβ2 0.80 0.27 0.83 2.17
    rβ3 0.37 0.24 0.92 2.21
    下载: 导出CSV
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  • 收稿日期:  2020-02-07
  • 刊出日期:  2020-06-25

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