人源IFITM2基因检测方法的建立及EMCV感染对IFITM2基因转录的影响  

Establishment of Human IFITM2 Gene Detection Method and Effect of EMCV Infection on IFITM2 Gene Transcription

作　　者：韩玉梅 毕英杰[1,2] 谢晶莹 许淑娟[1,2] 邓盈盈 张海霞 李向茸[1] 冯若飞 HAN Yu-mei;BI Ying-jie;XIE Jing-ying;XU Shu-juan;DENG Ying-ying;ZHANG Hai-xia;LI Xiang-rong;FENG Ruo-fei(Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;Life Science and Engineering College of Northwest MinzuUniversity,Lanzhou 730030,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[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,甘肃兰州730030 [2]西北民族大学生命科学与工程学院,甘肃兰州730030 [3]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《西北民族大学学报（自然科学版）》2020年第3期27-34,共8页Journal of Northwest Minzu University(Natural Science)

基　　金：国家自然科学基金项目(31460665);2019年中央专项研究生科研创新项目(Yxm2019148)。

摘　　要：目的:建立一种干扰素诱导跨膜蛋白2(IFITM2)基因SYBR Green I实时荧光定量PCR检测方法,以期为IFITM2基因的动态监测提供有效的检测技术.方法:根据GenBank中公布的IFITM2基因序列设计特异性引物,以质粒pMD18-T-IFITM2为模板进行条件优化,绘制标准曲线,建立IFITM2基因SYBR Green I实时荧光定量PCR检测方法,并对建立的方法进行条件优化与评价,用建立的方法对脑心肌炎病毒感染后细胞中IFITM2基因转录水平进行检测.结果:建立的20μL检测体系中IFITM2基因最佳引物浓度为10μM,最佳退火温度为59℃;标准质粒在2.62×105~2.62×1010 copies/μL浓度范围内与Ct值呈良好线性关系,线性方程y=-3.495 log x+43.12,R 2=0.998,且灵敏性、特异性和重复性均较高.应用建立的方法对EMCV感染细胞中IFITM2基因转录水平进行检测,结果显示,在感染早期IFITM2转录水平不变,感染后期转录水平逐渐升高.EMCV感染引起宿主IFITM2基因转录水平的变化,可能与EMCV逃逸宿主相关免疫应答有关.结论:成功建立了一种快速、准确检测IFITM2基因的SYBR Green I实时荧光定量PCR方法.该方法的建立,为进一步研究IFITM2在EMCV感染过程中的作用奠定了基础.Objective In order to establish a sensitive,specific and rapid detection method for interferon induced transmembrane protein 2(IFITM2),and provide an effective method for the detection of IFITM2 gene.Methods According to the sequence of IFITM2 gene published in GenBank,the specific primers were designed and the conditions were optimized by using plasmid pMD18-T-IFITM2 as template.Then drew the standard curve and established a SYBR Green I Real-time quantitative PCR method for the detection of IFITM2 gene.And the sensitivity,reproducibility and specificity of the method were verified.HEK293 cells which were infected with Encephalomyocarditis virus(EMCV),the expression of IFITM2 in cells was detected at the different times post infection.Results In the established 20μL detection system,the optimum primer concentration of IFITM2 gene was 10μM and the optimum annealing temperature was 59℃.The standard plasmid showed a good linear relationship with Ct value in the concentration range from 2.62×105 to 2.62×10 copies/μL,and the linear equation was y=-3.495 log x+43.12,R 2=0.998,and the sensitivity,specificity and repeatability were good.The transcription level of IFITM2 remained unchanged in the early stage of EMCV infection,but increased gradually in the later stage of infection.EMCV infection induced the change of IFITM2 gene transcription level in the host,which may be related to the immune evasion related to EMCV infection.Conclusion A rapid and accurate method for quantification of IFITM2 gene by SYBR Green I real-time quantitative PCR was successfully developed,which laid a foundation for further study of the role of IFITM2 in the infection of EMCV.

关 键 词：人干扰素诱导跨膜蛋白2 SYBR Green I实时荧光定量PCR 脑心肌炎病毒 

分 类 号：R-332[医药卫生]