稳定表达人IFITM3的A549细胞株建立及其对禽流感病毒感染的抑制作用  

Establishment of A549 cell stain stably expressing human IFITM3 and its inhibition to avian influenza virus infection

作　　者：姜颖越 杜寿文[1,2] 曹婷婷[1,2] 赵飞[1,2] 王茂鹏[1,2] 谭鹏[1,2] 辛舒[1,2] 朱羿龙[1,2] 李文杰[1,2] 李艳茹[1] 李太元[1] 李昌[1,2,3] 金宁一[1,2,3] 

机构地区：[1]延边大学农学院,延吉133002 [2]吉林省人兽共患病预防与控制重点实验室,军事医学科学院军事兽医研究所,长春130122 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国免疫学杂志》2016年第3期372-376,381,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(31472197);吉林省青年科研基金项目(20140520173JH);病原微生物生物安全国家重点实验室开放课题(SKLPBS1435)资助

摘　　要：目的:为深入研究IFITM3抗禽流感病毒作用及其分子机制提供细胞模型,建立稳定表达人IFITM3蛋白的A549细胞株。方法:首先合成引物,分离人外周血单个核细胞,PCR扩增IFITM3基因,进行DNA测序分析。测序正确后,将其克隆至慢病毒表达载体p LV-Puro中构建真核表达质粒p LV-IFITM3,转染A549细胞,在确定IFITM3可表达后,利用嘌呤霉素加压筛选稳定表达IFITM3的细胞株,通过荧光定量PCR和Western blot方法对获得的稳定细胞株进行鉴定,同时分析IFITM3蛋白在稳定细胞株的分布及其对细胞活性的影响。最后,基于建立的稳定细胞株,用H5N1亚型禽流感病毒作为病毒测试模型,对IFITM3的抗病毒活性进行了初步评价。结果:成功获得人外周血单个核细胞源IFITM3基因,构建了重组慢病毒表达质粒,并通过质粒转染结合嘌呤霉素压力筛选获得稳定表达IFITM3的A549细胞,且IFITM3主要分布在膜组分上,并对细胞活性无显著影响,同时IFITM3可抑制H5N1亚型禽流感病毒感染。结论:成功获得稳定表达IFITM3的A549细胞,证明了IFITM3可抑制H5N1亚型禽流感病毒感染,为进一步深入探讨IFITM3限制流感病毒感染的作用机制和分子机理提供了细胞模型和实验基础。Objective： To establish human lung carcinoma A549 cell stain stably expressing human interferon-inducible transmembrane protein 3（ IFITM3） and provide a cell model for the research of the antiviral effect of IFITM3 and its molecular mechanism. Methods： Full-length IFITM3 gene was amplified from the c DNA of human peripheral blood mononuclear cells isolated previously,and cloned into a lentivirus expression vector p LV-Puro resulting into p LV-IFITM3 after DNA sequencing. IFITM3 was transfected into A549 cells to screen the cell stain stably expressing IFITM3（ A549-IFITM3） with puromycin. The expression and distribution of IFITM3 and cell viability were detected and by quantitative PCR,Western blot and MTS assay respectively. Then the inhibitory effect of IFITM3 protein was preliminarily evaluated with H5N1 avian influenza virus as the virus model. Results： The A549 cell stain stably expressing IFITM3 was obtained by plasmid transfection and puromycin screening,and IFITM3 mainly distributed in the membrane fraction in the cell strain and had no significant effect on cell viability,and displayed strong restriction effect on H5N1 infection. Conclusion： A549 cells stably expressing IFITM3 were established and IFITM3 restricted avian influenza virus infection,providing the cell model and experimental basis to further explore its antiviral role and the molecular mechanism.

关 键 词：干扰素诱导跨膜蛋白3 稳定表达 禽流感病毒 抑制作用 

分 类 号：S855.3[农业科学—临床兽医学]