| TRIM21基因SYBR GreenⅠ实时荧光定量PCR检测方法的建立 |
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| 引用本文： | 赵克学,许淑娟,马瑞仙,耿金静,刘杨,李琼毅,冯若飞.TRIM21基因SYBR GreenⅠ实时荧光定量PCR检测方法的建立[J].热带生物学报,2020,11(1):111-117. |
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| 作者姓名： | 赵克学 许淑娟 马瑞仙 耿金静 刘杨 李琼毅 冯若飞 |
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| 作者单位： | 1.西北民族大学 生物医学研究中心/生物工程与技术国家民委重点实验室，兰州 730030 |
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| 基金项目： | 国家自然科学基金项目（31702234，31460665）；中央专项研究生科研创新项目（Yxm2018146）；教育部“创新团队发展计划”项目（IRT_17R88）；引进人才科研启动项目（xbmuyjrc201627）；中央高校项目（31920180123） |
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| 摘 要： | 三基序蛋白21（TRIM21）与癌症、肿瘤及固有免疫等密切相关。为了快速检测TRIM21基因的表达，根据Genbank中公布的TRIM21基因序列设计引物，通过常规PCR扩增该基因后克隆至pMD-18T载体上，测序并鉴定正确后制备重组质粒，以pMD-18T-TRIM21重组质粒作为标准品进行荧光定量PCR并建立标准曲线。采用重复性、特异性及灵敏性等试验，成功建立了检测TRIM21基因的荧光定量PCR检测方法。结果显示，该方法灵敏度高，最低检测下限为37.7 copies·μL?1，比普通PCR高出10倍。在3次独立的试验中，组内和组间变异系数较小，具有较高的可重复性，同时，还能检出不同细胞中TRIM21的表达。因此，该荧光定量方法可为TRIM21的检测及临床研究提供技术支持。
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| 关 键 词： | 三基序蛋白21 荧光定量PCR 标准曲线 |
| 收稿时间： | 2019-09-12 |
Establishment of SYBR Green I Real-time PCR for Quantitative Detection of TRIM21 Gene |
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| Institution: | 1.Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission/Biomedical Research Center, Northwest Minzu University, Lanzhou 7300302.Life Science and Engineering College, Northwest Minzu University, Lanzhou, Gansu 730030, China |
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| Abstract: | TRIM21 is closely associated with cancer, tumor, and innate immunity, etc. In order to detect TRIM21 gene expression quickly, primers were designed and synthesized according to the sequence of TRIM21 gene in the Genbank. The TRIM21 gene was amplified by conventional PCR and cloned into pMD-18T vector.After sequencing and identification, the recombinant plasmid was prepared and used for SYBR Green I real-time PCR to establish standard curves, based on which the PCR method was successfully established after repetition, specificity and sensitivity tests. This method was high in sensitivity, and its minimum detection limit was 37.7 copies·μL?1, which was 10 times higher than that of the conventional PCR. Three separate repetition tests of this method showed that the inter-assay and intra-assay repetitions had a lower coefficient of variation, less than 5%, indicating a high repetition. Meanwhile, TRIM21 expression was also detected in different cells. Therefore, the TRIM21 gene SYBR Green I real-time PCR method establishedshould behelpful for the detection of TRIM21 gene expression and clinical research. |
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