稳定表达PRRSV N蛋白的Marc-145细胞系的构建  

Construction of a Marc-145 Cell Line Stably Expressing PRRSV N Protein

作　　者：任同伟 覃念[1] 全东群 谢欣 王豪 王玉旭 陈樱[1] 欧阳康[1] 黄伟坚[1] 韦祖樟[1] REN Tongwei;QIN Nian;QUAN Dongqun;XIE Xin;WANG Hao;WANG Yuxu;CHEN Ying;OUYANG Kang;HUANG Weijian;WEI Zuzhang(Laboratory of Animal infectious Diseases and molecular Immunology,College of Animal Science and Technology,Guangxi University,Nanning 530005,China)

机构地区：[1]广西大学动物科学技术学院动物传染病与分子免疫学实验室,南宁530005

出　　处：《中国动物传染病学报》2022年第1期15-19,共5页Chinese Journal of Animal Infectious Diseases

基　　金：广西自然科学基金(2016GXNSFAA380159,2018GXNSFDA281021);国家自然科学基金(31660716)。

摘　　要：为了构建稳定表达猪繁殖与呼吸综合征病毒(PRRSV)N蛋白的Marc-145细胞系,以PRRSV全长感染性克隆为模板,通过PCR方法扩增PRRSV N基因,将N基因克隆到慢病毒载体中,获得重组质粒pLenti-CMV-N,利用三质粒慢病毒包装系统转染293T细胞,包装成表达N蛋白的慢病毒颗粒,将慢病毒颗粒转导至Marc-145细胞,用潮霉素B初步筛选阳性细胞,采用终点稀释的方法筛选出稳定表达PRRSV N蛋白的Marc-145细胞系。通过的PCR扩增N基因和测序表明细胞系基因组中存在N蛋白编码序列,IFA和Western blot试验验证了N蛋白在细胞系中稳定表达。为此,我们成功获得了稳定表达PRRSV N蛋白的Marc-145细胞系。In order to construct a Marc-145 cell line stably expressing PRRSV N protein,the N gene was amplifi ed by PCR using an infectious cDNA clone(pGXAM)as template.Then the N gene fragment was cloned into Lentiviral vector,resulting a recombinant clone pLenti-CMV-N.Three-plasmid Lentiviral packaging system containing N gene was then transfected into 293T cells.The packaged lentiviral particles were transducted into Marc-145 cells.The Marc-145 cell lines stably expressing PRRSV N protein were screened by hygromycin B and end-point dilution.Sequencing of PCR products showed the genome of the transducted Marc-145 cell line contained N gene.The expression of the N protein in this cell line was confi rmed by IFA and Western blot.These results showed that a Marc-145 cell line was constructed for stable expression of PRRSV N protein.

关 键 词：PRRSV N蛋白 细胞系 慢病毒载体 

分 类 号：S852.659.6[农业科学—基础兽医学]