长链非编码RNA(lncRNA)-MSTRG.22610.1对BHV-1体外复制影响的研究  

The effect of lncRNA-MSTRG.22610.1 on the proliferation of BHV-1 virus in vitro

作　　者：姜坤生 王禹淳 马金柱[1] 于立权[1] 宋佰芬[1,2] JIANG Kun-sheng;WANG Yu-chun;MA Jin-zhu;YU Li-quan;SONG Bai-fen(College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]中国农业大学动物医学院,北京100193

出　　处：《中国预防兽医学报》2024年第3期278-284,299,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然基金面上项目(32272991);中国农业大学“小组团”援疆团队项目。

摘　　要：本研究室前期将牛疱疹病毒Ⅰ型(BHV-1)感染牛肾细胞(MDBK),通过转录组测序筛选到了转录显著差异的长链非编码RNA-MSTRG.22610.1(lncRNA-MSTRG.22610.1),为了探究其在MDBK中对BHV-1复制的影响,本研究采用CCK-8法筛选对细胞无毒性的聚凝胺、嘌呤霉素的最佳浓度;将重组慢病毒r ZHLV-U6-Zs Green1-puro-MSTRG.22610.1和r ZHLV-U6-Zs Green1-puro(对照慢病毒)按照不同的MOI分别感染MDBK细胞,48 h后观察荧光,采用Image J软件检测并计算各慢病毒感染细胞后出现绿色荧光细胞的比率,出现绿色荧光细胞的比率达80%的细胞对应的MOI即为最佳MOI。筛选结果显示聚凝胺与嘌呤霉素的最佳工作浓度分别为5μg/mL及3μg/m L,慢病毒感染的最适MOI为80。将r ZHLV-U6-Zs Green1-puro-MSTRG.22610.1和对照慢病毒分别以最佳条件感染MDBK细胞并培养及传代,传至8~10代,每代均观察细胞形态及细胞中的绿色荧光,并通过RT-q PCR检测第6、8、10代细胞中lncRNA-MSTRG.22610.1的转录水平,以构建并鉴定稳定表达lncRNA-MSTRG.22610.1的MDBK细胞系1T及对照细胞系1NC。结果显示,每代1T细胞、1NC细胞的状态均良好并均与阴性对照MDBK细胞的形态无差别,且每代1T及1NC细胞均出现绿色荧光。RT-q PCR结果显示,与1NC及阴性对照细胞相比,1T细胞系中lncRNA-MSTRG.22610.1的转录水平极显著升高(P<0.0001)。表明获得了高水平表达lncRNA-MSTRG.22610.1却不影响细胞增殖的细胞系,且该细胞系的遗传稳定性较强。将BHV-110倍倍比稀释后分别感染传至10代的1T与1NC细胞,24 h后采用Reed-Muench法计算各组细胞中的病毒滴度。结果显示,1T、1NC及MB细胞(感染BHV-1的MDBK细胞)的病毒滴度分别为105.15 TCID50/0.1 m L、104.41TCID50/0.1 m L及104.58 TCID50/0.1 m L。与MB和1NC细胞相比,1T细胞中的病毒滴度显著升高(P<0.05),表明lnc RNA-MSTRG.22610.1表达后促进BHV-1在MDBK细胞中的复制。本研究为进一步探究lncRNA-MSTRG.22610.1促进病�Transcriptome sequencing of Bovine kidney cells(MDBK)pre-infected with bovine herpesvirus type I(BHV-1)revealed a long-stranded non-coding RNA-MSTRG.22610.1(lncRNA-MSTRG.22610.1)with significant differences in transcription.In order to investigate the effect of RNA-MSTRG.22610.1 on BHV-1 replication in MDBK cells,this study used the CCK-8 method to screen the optimal concentrations of polyglutamine and puromycin,which are non-toxic to cells.Recombinant lentiviruses r ZHLV-U6-ZsGreen1-puro-MSTRG.22610.1 and r ZHLV-U6-ZsGreen1-puro(control lentiviruses)were used to infect MDBK cells with different MOIs,and fluorescence was observed 48 hours after infection.The fluorescence of each lentivirus was detected,and the percentage of green fluorescent cells was calculated by Image J software for each lentivirus-infected cell.The MOI corresponding to the cell with 80%of green fluorescent cells was the best MOI.The screening results showed that the optimal working concentration of polyglutamine was 5μg/m L,the optimal working concentration of puromycin was 3μg/m L,and the optimal MOI for lentiviral infection was 80.To construct and characterize the MDBK cell line 1T stably expressing lncRNA-MSTRG.22610.1 and the control cell line 1NC,MDBK cells infected with r ZHLV-U6-ZsGreen1-puro-MSTRG.22610.1and the control lentiviruses with optimal infection MOIs,respectively.The cells were cultured for 8-10 passages,and the cell morphology and green fluorescence in the cells were observed in each passage.RT-qPCR detected the transcript levels of lncRNA-MSTRG.22610.1 in the cells of the 6th,8th,and 10th passages.The results showed that each passage of 1T cells and1NC cells was in good condition and had no difference in morphology with the negative control MDBK cells,and each generation of 1T and 1NC cells showed green fluorescence.RT-qPCR results showed that the transcription level of lncRNA-MSTRG.22610.1in 1T cell line was significantly higher than in 1NC and negative control cells(P<0.0001).The results showed that a cell line with hi

关 键 词：长链非编码RNA 牛肾细胞 牛疱疹病毒I型 慢病毒过表达 病毒增殖 

分 类 号：S852.65[农业科学—基础兽医学]