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CHRNA7基因荧光定量PCR检测方法的建立

刘益巧,  孙志华,  钱江,  长孙东亭,  罗素兰

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文章导航 > 热带生物学报  > 2019 >  10(2): 178-183
刘益巧, 孙志华, 钱江, 长孙东亭, 罗素兰. CHRNA7基因荧光定量PCR检测方法的建立[J]. 热带生物学报, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013
引用本文: 刘益巧, 孙志华, 钱江, 长孙东亭, 罗素兰. CHRNA7基因荧光定量PCR检测方法的建立[J]. 热带生物学报, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013
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LIU Yiqiao, SUN Zhihua, QIAN Jiang, ZHANGSUN Dongting, LUO Sulan. Establishment of Real-time PCR for Detection of CHRNA7 Gene[J]. Journal of Tropical Biology, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013
Citation: LIU Yiqiao, SUN Zhihua, QIAN Jiang, ZHANGSUN Dongting, LUO Sulan. Establishment of Real-time PCR for Detection of CHRNA7 Gene[J]. Journal of Tropical Biology, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013
shu

CHRNA7基因荧光定量PCR检测方法的建立

doi: 10.15886/j.cnki.rdswxb.2019.02.013

  • 海南大学 海洋学院/海南大学热带生物资源教育部重点实验室/海口市海洋药物重点实验室, 海口 570228

基金项目: 

国家自然科学基金项目(81872794)

详细信息
    第一作者:

    刘益巧(1994-),女,海南大学海洋学院2016级硕士研究生,E-mail:576494486@qq.com

    通信作者:

    罗素兰(1969-),女,教授,博导.研究方向:海洋药物与生物技术,E-mail:luosulan2003@163.com

  • 中图分类号: Q786

Establishment of Real-time PCR for Detection of CHRNA7 Gene

  • College of Marine, Hainan University/Key Laboratory of Tropical Biological Resources of Ministry of Education/Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China

  • 摘要: 为了检测烟碱型乙酰胆碱受体（nicotine acetylcholine receptors,nAChRs）α7亚基基因（CHRNA7）的表达,笔者根据人源CHRNA7基因序列设计引物,PCR扩增CHRNA7基因,亚克隆到pMD-18T载体中后,测序鉴定并制备重组质粒,以梯度稀释的重组质粒为模板,进行荧光定量PCR来绘制标准曲线,并进行灵敏度和重复性实验,成功建立了检测CHRNA7基因的荧光定量PCR方法。该方法最低可检测101 copies·μL-1,且重复性良好,在10～4,105,106 copies·μL-1时,5次重复的变异系数分别是1.22%,1.90%,2.63%。此外,还对人宫颈癌细胞SiHa和人正常宫颈细胞Ect1/E6E7中α7 nAChR亚基基因表达量进行检测,结果显示,α7 nAChR亚基在SiHa细胞系的表达明显低于其在Ect1/E6E7细胞系中的表达（P=0.015）。
    Abstract: In order to detect the expression of α7 nicotine acetylcholine receptor（nAChR） subunit gene（CHRNA7）, the primers were designed according to the sequence of human CHRNA7 gene, and then CHRNA7 gene was amplified by PCR. After being cloned into pMD-18 T vector, the recombinant plasmid was sequenced and prepared. The recombinant plasmids were gradient diluted and used as templates to establish standard curves for real-time PCR. The sensitivity and repeatability of the real-time PCR were carried out. A real-time PCR method for detecting CHRNA7 was successfully established. With this method CHRNA7 gene was detected 10 copies·μL-1 at the lowest. The variation coefficients of five replications of the three templates at 10~4, 105 and 106 copies·μL-1 were 1.22%, 1.90% and 2.63%, respectively, which indicated the real-time PCR detection had a good repeatability. Furthermore, the detection of α7 nAChR subunit gene expression in human cervical cancer cell line SiHa and human normal cervical cell line Ect1/E6 E7 showed that the expression of α7 nAChR subunit in SiHa cells was significantly lower than that in Ect1/E6 E7 cells（P=0.015）.
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    • 收稿日期:  2019-03-25
    • 修回日期:  2019-04-05

    CHRNA7基因荧光定量PCR检测方法的建立

    doi: 10.15886/j.cnki.rdswxb.2019.02.013
    • 海南大学 海洋学院/海南大学热带生物资源教育部重点实验室/海口市海洋药物重点实验室, 海口 570228
      • 基金项目:

      国家自然科学基金项目(81872794)

      作者简介:

      刘益巧(1994-),女,海南大学海洋学院2016级硕士研究生,E-mail:576494486@qq.com

      通讯作者: 罗素兰(1969-),女,教授,博导.研究方向:海洋药物与生物技术,E-mail:luosulan2003@163.com
    • 收稿日期: 2019-03-25
    • 修回日期: 2019-04-05

    • 中图分类号: Q786

    摘要: 为了检测烟碱型乙酰胆碱受体（nicotine acetylcholine receptors,nAChRs）α7亚基基因（CHRNA7）的表达,笔者根据人源CHRNA7基因序列设计引物,PCR扩增CHRNA7基因,亚克隆到pMD-18T载体中后,测序鉴定并制备重组质粒,以梯度稀释的重组质粒为模板,进行荧光定量PCR来绘制标准曲线,并进行灵敏度和重复性实验,成功建立了检测CHRNA7基因的荧光定量PCR方法。该方法最低可检测101 copies·μL-1,且重复性良好,在10～4,105,106 copies·μL-1时,5次重复的变异系数分别是1.22%,1.90%,2.63%。此外,还对人宫颈癌细胞SiHa和人正常宫颈细胞Ect1/E6E7中α7 nAChR亚基基因表达量进行检测,结果显示,α7 nAChR亚基在SiHa细胞系的表达明显低于其在Ect1/E6E7细胞系中的表达（P=0.015）。

    English Abstract

    刘益巧, 孙志华, 钱江, 长孙东亭, 罗素兰. CHRNA7基因荧光定量PCR检测方法的建立[J]. 热带生物学报, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013
    引用本文: 刘益巧, 孙志华, 钱江, 长孙东亭, 罗素兰. CHRNA7基因荧光定量PCR检测方法的建立[J]. 热带生物学报, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013
    shu
    LIU Yiqiao, SUN Zhihua, QIAN Jiang, ZHANGSUN Dongting, LUO Sulan. Establishment of Real-time PCR for Detection of CHRNA7 Gene[J]. Journal of Tropical Biology, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013
    Citation: LIU Yiqiao, SUN Zhihua, QIAN Jiang, ZHANGSUN Dongting, LUO Sulan. Establishment of Real-time PCR for Detection of CHRNA7 Gene[J]. Journal of Tropical Biology, 2019, 10(2): 178-183. doi: 10.15886/j.cnki.rdswxb.2019.02.013
    shu

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