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医学临床研究  2018, Vol. 35 Issue (10): 1886-1888    DOI: 10.3969/j.issn.1671-7171.2018.10.005
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TNFAIP1-shRNA慢病毒表达载体的构建与鉴定
丰昀1, 彭文婷2, 张嘉玥2, 张雅倩2, 张敏3, 王光伟3
1.长沙市中心医院眼科,湖南 长沙 410004;
2.湖南医药学院临床医学院,湖南 怀化 418000;
3.湖南医药学院生物医学研究中心,湖南 怀化 418000
Construction and Identification of TNFAIP1-shRNA lentiviral Expression Vector
FENG Yun, PENG Wen-ting, ZHANG Jia-yue, et al
Department of Ophthalmology, Changsha Central Hospital, Changsha 410004, China
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摘要 【目的】构建肿瘤坏死因子-α诱导蛋白-1 (TNFAIP1)基因短发卡RNA (shRNA)慢病毒载体。【方法】根据人TNFAIP1基因序列设计合成4条shRNA,构建shRNA表达质粒,将重组质粒转染至293T细胞,应用RT-PCR检测各组TNFAIP1基因mRNA的表达水平,筛选出最有效的干扰靶序列,构建HSH018143-LVRH1P-RNAi慢病毒载体,并进行测序鉴定。将确定了的TNFAIP1-shRNA-0S523409转染至293T细胞,72 h后,观察绿色荧光表达情况,并测定病毒滴度。【结果】TNFAIP1-shRNA慢病毒表达载体高效转导入293T细胞并稳定表达,测定其病毒滴度为5×108 TU/mL。【结论】本研究成功构建了TNFAIP1基因shRNA慢病毒表达载体,为研究TNFAIP1在糖尿病视网膜病变中的作用奠定了实验基础。
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Abstract:【Objective】To construct tumor necrosis factor alpha-induced protein 1 (TNFAIP1)-shRNA lentiviral vector.【Methods】Four shRNA sequences were designed and synthesized according to the sequence of human TNFAIP1 gene. These recombinant plasmids were transfected into 293T cells. RT-PCR assay was applied to detect the mRNA expression level of TNFAIP1 in each group and thus to choose the most effective TNFAIP1-shRNA. The HSH018143-LVRH1P-RNAi was constructed and confirmed by DNA sequencing. The selected TNFAIP1-shRNA-0S523409 was transfected into 293T cells. After 72 hours, the green fluorescence expression was observed and the lentiviral titer was tested.【Results】The TNFAIP1-shRNA lentiviral vector had been transfected into 293T cells efficiently and stably, the titer of lentivirus was 5×108 TU/mL.【Conclusion】The TNFAIP1-shRNA lentiviral expression vector has been constructed successfully, which lays a foundation for exploring for the role of TNFAIP1 in diabetic retinopathy.
收稿日期: 2018-02-28     
PACS:  R373  
基金资助:湖南省科技计划项目(No.2015TP1020);怀化市科技计划重点项目(No. 2017X2102);湖南省教育厅一般项目(No. 17C1154);2017年度湖南省大学生研究性学习和创新性实验计划项目(No. 945)
通讯作者: E-mail:wanggwwmq323@163.com   
引用本文:   
丰昀, 彭文婷, 张嘉玥, 张雅倩, 张敏, 王光伟. TNFAIP1-shRNA慢病毒表达载体的构建与鉴定[J]. 医学临床研究, 2018, 35(10): 1886-1888.
FENG Yun, PENG Wen-ting, ZHANG Jia-yue, et al. Construction and Identification of TNFAIP1-shRNA lentiviral Expression Vector. JOURNAL OF CLINICAL RESEARCH, 2018, 35(10): 1886-1888.
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