HeLa细胞Ⅰ型干扰素效应因子实时荧光定量 RT-PCR检测方法的建立及初步应用

›› 2013, Vol. ›› Issue (5): 1-7.

• 生物技术 • 下一篇

HeLa细胞Ⅰ型干扰素效应因子实时荧光定量 RT-PCR检测方法的建立及初步应用

刘纪玉^1,2, 杜以军^2,3, 刘星², 吴家强^2,3, 李俊^2,3, 丛晓燕^2,3, 赵新华^2,3, 徐绍建^2,3, 孙文博^2,3, 时建立^2,3, 单虎¹, 王金宝^1,2,3

1. 青岛农业大学, 山东省预防兽医学重点实验室, 山东青岛 266109;
2. 山东省畜禽疫病防治与繁育重点实验室, 山东济南 250100;
3. 山东省农业科学院畜牧兽医研究所, 山东济南 250100

收稿日期:2012-10-22 出版日期:2013-05-20 发布日期:2013-05-27
通讯作者: 单虎,教授。E-mail:shanhu67@163.com;王金宝,教授。E-mail:wangjb@saas.ac.cn E-mail:shanhu67@163.com;wangjb@saas.ac.cn
作者简介:刘纪玉(1986-),男,山东人,硕士生,研究方向:动物传染病与生物制品。
基金资助:
国家自然科学基金(31100119、31170146);青岛市科技基金(11-2-3-24-nsh);山东省生猪产业创新团队建设;公益性行业(农业)科研专项经费(201303046);山东省优秀中青年科学家科研奖励基金(BS2012NY012)。

Establishment and Preliminary Application of a Real-time Fluorescent Quantitative RT-PCR Assay for Detection of Type Ⅰ Interferon Effect Factor of HeLa Cells

LIU Ji-yu^1,2, DU Yi-jun^2,3, LIU Xing², WU Jia-qiang^2,3, LI Jun^2,3, CONG Xiao-yan^2,3, ZHAO Xin-hua^2,3, XU Shao-jian^2,3, SUN Wen-bo^2,3, SHI Jian-li^2,3, SHAN Hu¹, WANG Jin-bao^1,2,3

1. Shandong Key Laboratory of Preventive Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China;
2. Shandong Key Laboratory of Animal Disease Control and Breeding, Jinan 250100, China;
3. Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China

Received:2012-10-22 Online:2013-05-20 Published:2013-05-27

摘要/Abstract

摘要： 本试验旨在建立一种用SYBR Green Ⅰ荧光染料检测HeLa细胞Ⅰ型干扰素效应因子ISG15、ISG56、Mx1、OAS和PKR mRNA表达水平的实时荧光定量RT-PCR检测方法,并在口蹄疫病毒(FMDV)L蛋白抑制Ⅰ型IFN发挥效应的信号通路中进行初步应用。利用TRIzol法提取总RNA,经Oligo d(T)₁₅进行反转录,利用PCR扩增各段目的基因,并克隆至pMD18-T载体,转化大肠杆菌DH5α,经鉴定为阳性的重组质粒作为标准品模板建立SYBR Green Ⅰ荧光定量RT-PCR标准曲线和熔解曲线,并进行灵敏性、特异性和重复性试验。根据建立的实时荧光定量RT-PCR方法,检测FMDV L蛋白对Ⅰ型IFN效应因子的抑制效果。HeLa细胞在转染FMDV L蛋白真核表达质粒,并受到Ⅰ型IFN刺激后ISG15、ISG56、Mx1、OAS和PKR的相对表达量较转染空载体或表达GST的真核表达质粒明显降低。本试验建立了HeLa细胞Ⅰ型IFN效应因子的实时荧光定量RT-PCR检测方法,为在mRNA水平上对HeLa细胞Ⅰ型IFN效应因子的定量分析奠定了基础,并成功地初步应用于FMDV L蛋白抑制Ⅰ型IFN发挥效应的信号通路的研究中。

关键词: 实时荧光定量RT-PCR; SYBR Green Ⅰ; 口蹄疫病毒; L蛋白; 干扰素

Abstract: The assay was aimed to establish a SYBR Green Ⅰ fluorescent quantitative RT-PCR method to detect the mRNA of type Ⅰ interferon effect factor ISG15, ISG56, Mx1, OAS and PKR in HeLa cells. Using this method, foot and mouth disease virus (FMDV) L protein inhibiting the type Ⅰ IFN signaling pathway was evaluated. Total RNA was extracted by TRIzol reagent and Oligo d(T)₁₅ was used for reverse transcription, the target gene was amplified individually by PCR and cloned into pMD18-T vector, the positive recombinant plasmid was constructed and used as template for SYBR Green Ⅰ fluorescence quantitative RT-PCR. The standard curve and melting curve were established and the sensitivity, specificity and reproducibility assay were conducted. Using this established method, the inhibiting effect on type Ⅰ IFN effect factor of FMDV L protein was detected. The ISG15, ISG56, Mx1, OAS the PKR relative mRNA level of HeLa cells transfected with eukaryotic expression plasmid expressing FMDV L protein were obviously reduced compare to HeLa cells transfected with empty vector or eukaryotic expression plasmid expressing GST. The SYBR Green Ⅰ fluorescent quantitative RT-PCR method was established, which laid the foundation to evaluate the mRNA levels of type Ⅰ IFN effect factor in HeLa cells.This method was successfully applied to FMDV L protein inhibition of type Ⅰ IFN signaling pathway.

Key words: Real-time fluorescent quantitative RT-PCR; SYBR GreenⅠ; foot and mouth disease virus; L protein; interferon

中图分类号:

刘纪玉, 杜以军, 刘星, 吴家强, 李俊, 丛晓燕, 赵新华, 徐绍建, 孙文博, 时建立, 单虎, 王金宝. HeLa细胞Ⅰ型干扰素效应因子实时荧光定量 RT-PCR检测方法的建立及初步应用[J]. , 2013, (5): 1-7.

LIU Ji-yu, DU Yi-jun, LIU Xing, WU Jia-qiang, LI Jun, CONG Xiao-yan, ZHAO Xin-hua, XU Shao-jian, SUN Wen-bo, SHI Jian-li, SHAN Hu, WANG Jin-bao. Establishment and Preliminary Application of a Real-time Fluorescent Quantitative RT-PCR Assay for Detection of Type Ⅰ Interferon Effect Factor of HeLa Cells[J]. China Animal Husbandry & Veterinary Medicine, 2013, (5): 1-7.

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HeLa细胞Ⅰ型干扰素效应因子实时荧光定量 RT-PCR检测方法的建立及初步应用

Establishment and Preliminary Application of a Real-time Fluorescent Quantitative RT-PCR Assay for Detection of Type Ⅰ Interferon Effect Factor of HeLa Cells

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