干扰素受体1沉默的人二倍体MRC-5细胞系对水痘-带状疱疹病毒复制的影响  

Effect of interferon receptor 1 silenced human diploid MRC-5 cell line on replication of varicella-zoster virus

作　　者：杨骁 姜承瀚 孙博 谷铁军 万明明 孙捷 丁雪 王岑嵘 周恩同 姜皓[3] 苏维恒 YANG Xiao;JIANG Cheng-han;SUN Bo;GU Tie-jun;WAN Ming-ming;SUN Jie;DING Xue;WANG Cen-rong;ZHOU En-tong;JIANG Hao;SU Wei-heng(National Engineering Laboratory for AIDS Vaccine,College of Life Science,Jilin University,Changchun 130012,Jilin Province,China;不详)

机构地区：[1]吉林大学生命科学学院艾滋病疫苗国家工程实验室,吉林长春130012 [2]延边大学农学院园艺园林系,吉林延边朝鲜族自治州133002 [3]吉林大学中日联谊医院手术室,吉林长春130033

出　　处：《中国生物制品学杂志》2023年第1期21-25,31,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(32170944,32070932,31770184);吉林省科技发展计划基金(20220402044GH);内蒙古自治区科技攻关计划(2020GG0233)。

摘　　要：目的通过优化人二倍体细胞系MRC-5降低其干扰素(interferon,IFN)相关基因表达水平,以提高水痘-带状疱疹病毒(varicella-zoster virus,VZV)在该细胞系中的复制水平,并提高VZV疫苗产量。方法通过CRISPR/Cas9基因编辑技术,构建IFN受体1(interferon receptor 1,IFNAR1)沉默的MRC-5细胞系(MRC-5^(IFNAR1-))。采用qRT-PCR法检测MRC-5^(IFNAR1-)细胞系IFNAR1 mRNA相对表达量,同时检测VZV感染后IFN相关基因IFNβ和OAS1 mRNA相对表达量,评价基因沉默效果。通过沉默位点的基因测序进一步鉴定基因突变序列。采用qRT-PCR法和空斑形成单位(plaque formation unit,PFU)试验,对比VZV感染后168 h病毒在MRC-5和MRC-5^(IFNAR1-)细胞系中的复制情况,评价MRC-5^(IFNAR1-)细胞系对VZV复制的影响。结果MRC-5^(IFNAR1-)细胞系生长状态与MRC-5细胞一致,IFNAR1 mRNA相对表达量降低73%;VZV感染后MRC-5^(IFNAR1-)细胞系中IFN相关基因IFNβ和OAS1 mRNA相对表达量比MRC-5细胞分别降低了61%和90%;VZV感染后168 h,病毒DNA复制水平增加5.7倍,病毒滴度增加4倍。结论成功建立了MRC-5^(IFNAR1-)细胞系,可作为增加基于人二倍体细胞疫苗产量的潜在方案,为扩大VZV疫苗生产提供了参考。Objective To improve the replication level of varicella-zoster virus(VZV)in human diploid cell line MRC-5 and increase the yield of VZV vaccine by reducing the expression of interferon(IFN)related genes via optimizing the cell line MRC-5.Methods Interferon receptor 1(IFNAR1)silenced MRC-5 cell line(MRC-5^(IFNAR1-))was constructed by CRISPR/Cas9 gene editing technology,which was determined for the relative expression of IFNAR1 mRNA,and for those of mRNA of IFN related genes IFNβand OAS1 after VZV infection by qRT-PCR to evaluate the effect of gene silencing.Gene mutation sequences were further identified by sequencing of the silenced sites.The replication of VZV in MRC-5 and MRC-5^(IFNAR1-)cell lines was compared 168 h after VZV infection by using qRT-PCR and plaque formation unit(PFU)assay,to evaluate the effect of MRC-5^(IFNAR1-)cell line on VZV replication.Results The growth status of MRC-5^(IFNAR1-)cell line was consistent with that of MRC-5 cells,and the relative expression of IFNAR1 mRNA decreased by 73%;The relative expressions of IFNβand OAS1 mRNA in MRC-5^(IFNAR1-)cell line were 61%and 90%lower than those in MRC-5 cells respectively after VZV infection;In addition,168 h after VZV infection,the level of DNA replication and the titer of VZV increased by 5.7 folds and 4 folds respectively.Conclusion The successful establishment of MRC-5^(IFNAR1-)cell line may be a potential scheme to increase the yield of vaccines based on human diploid cells,and provided a reference for expanding production of VZV vaccine.

关 键 词：水痘-带状疱疹病毒 人二倍体MRC-5细胞 干扰素应答 基因沉默 

分 类 号：R392.11[医药卫生—免疫学]