稳定表达人TMPRSS2基因BHK细胞系的建立及其对新城疫弱毒增殖效率评价  

Establishment of BHK cell line stably expressing human TMPRSS2 gene and evaluation of lentogenic Newcastle disease virus proliferation efficiency

作　　者：刘伟[1] 李梦娇 郭佩东 孙英杰[2] 仇旭升[2] 谭磊[2] 宋翠萍[2] 廖瑛[2] 孟春春[2] 丁铲 LIU Wei;LI Meng-jiao;GUO Pei-dong;SUN Ying-jie;QIU Xu-sheng;TAN Lei;SONG Cui-ping;LIAO Ying;MENG Chun-chun;DING Chan(College of Animal Science(College of Apiology),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]福建农林大学动物科学学院(蜂学学院),福建福州350002 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医科学》2021年第5期594-600,共7页Chinese Veterinary Science

基　　金：上海市兽医生物技术重点实验室开放基金项目(Klab201702)。

摘　　要：为提高细胞培养系统中新城疫病毒的增殖效率,通过构建TMPRSS2过表达的BHK细胞系,探索其对新城疫弱毒增殖的影响。本研究将TMPRSS2基因克隆于噬菌体载体pHAGE中,构建重组质粒pHAGE-TMPRSS2。将重组质粒pHAGE-TMPRSS2以及辅助包装质粒psPAX2和pMD2G共转染至293T细胞并收集上清。将包装好的慢病毒感染BHK细胞,通过Blasticidin加入筛选和有限稀释法将单克隆细胞扩大培养。经RT-PCR和Western-blot鉴定,结果表明,TMPRSS2在BHK细胞中获得了稳定的高表达。接种新城疫弱毒后通过TCID50测定显示,TMPRSS2过表达BHK细胞系能支持较高水平的病毒增殖。In order to improve the proliferation efficiency of Newcastle disease virus(NDV) in cell culture system,the effect of TMPRSS2 over expression BHK cell line on the proliferation of lentogenic NDV was explored.In this study,TMPRSS2 gene was cloned into phage vector phage to construct recombinant plasmid p HAGE-TMPRSS2.The recombinant plasmid p HAGE-TMPRSS2,as well as the helper packaging plasmids ps PAX2 and p MD2 G were co-transfected into 293 T cells,then the supernatant was collected.BHK cells were infected with packaged lentivirus,and the monoclonal cells were expanded by blasticidin screening and limited dilution method. RT-PCR and Western-blot showed that TMPRSS2 was stably and highly expressed in BHK cells.After inoculation with lentogenic NDV,TCID50 assay showed that TMPRSS2 overexpression BHK cell line could support high level of virus proliferation.

关 键 词：TMPRSS2 慢病毒包装 BHK细胞 新城疫弱毒 

分 类 号：S852.659.5[农业科学—基础兽医学]