反转录病毒MSCV介导汉坦病毒核蛋白基因在NIH3T3细胞中整合和表达  

Retrovirus MSCV Mediated Integration and Expression of Hantavirus Nucleocapsid Protein in NIH 3T3 Cells

作　　者：刘合宾[1] 刘克洲[1] 翁景清[2] 朱智勇[2] 

机构地区：[1]浙江大学医学院附一医院传染病研究所,杭州310003 [2]浙江省疾病预防控制中心肾综合征出血热重点实验室,杭州310009

出　　处：《病毒学报》2004年第4期298-302,共5页Chinese Journal of Virology

基　　金：浙江省自然科学基金资助(项目编号:397417)

摘　　要：汉坦病毒是引起肾综合征出血热(HFRS)和汉坦病毒型肺炎综合征(HPS)的主要病原体。由S基因编码的核蛋白(NP)主要与机体的细胞免疫有关,并调节病毒的复制及诱导细胞程序性死亡。构建了汉坦病毒Z10株核蛋白cDNA与含有pac基因的反转录病毒鼠干细胞病毒(MSCV)重组体MSCV-FlagNP,通过磷酸钙转录法导入产病毒的包装细胞系BOSC23中,产生完整的重组MSCV-FlagNP病毒。然后以重组病毒感染NIH3T3细胞,利用Puromycin的选择特性(pac基因)对感染细胞进行连续压力筛选,获得了转核蛋白抗性细胞。利用Southernblot和PCR方法分别对核蛋白基因在抗性细胞染色体整合情况及其完整性进行了鉴定。并且用Westernblot在抗性细胞中可检测到核蛋白的表达。进一步以Flag单克隆抗体介导的免疫荧光染色联合共聚焦激光扫描荧光显微镜,分析了内源性Flag融合核蛋白在抗性细胞内分布,发现核蛋白主要分布于胞浆及胞核周围区,并且部分核蛋白可聚集形成胞浆包涵体。转核蛋白基因细胞模型的建立,对进一步研究汉坦病毒核蛋白功能以及病毒复制机制有重要意义。Hantaviruses are the major causative agents of human hemorrhagic fever with renal syndrome(HFRS)and hantavirus pulmonary syndrome(HPS).The viral nucleocapsid protein(NP),encoded by S gene,plays very important roles in regulation of cellular immune response,virus replication and apoptosis.In this study,the full length NP cDNA with Flag sequence was cloned into the retroviral vector MSCV containing puromycin resistant gene pac.By transfection of the resulting recombinant plasmid MSCV-FlagNP into the packaging cell BOSC23,the complete retrovirus MSCV-FlagNP was generated and used to infect NIH 3T3 cells.Then the retrovirus-transduced cells were obtained by constitutive puromycin selection.The integration of NP gene in the genomic DNA of selected cell clones was further confirmed by PCR and Southern blot.In addition,the expression of Flagtagged NP in transduced cells was analyzed by Western blot with anti-Flag monoclonal antibody.Furthermore,IFA in combination with laser scanning confocal fluorescent microscope showed the expressed NP predominantly located and partially dimerized into inclusion bodies in the cytoplasm of transduced cells.Our data suggest it was significant of establishing NP-transduced cells through retrovirus mediated integration to stably and constitutively express NP in transgenic cells.

关 键 词：核蛋白基因 汉坦病毒 胞浆 介导 表达 NIH 3T3细胞 ac基因 反转录病毒 利用 

分 类 号：Q813.11[生物学—生物工程] R373[医药卫生—病原生物学]