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
姜坤生 1王禹淳 1马金柱 1于立权 1宋佰芬2
作者信息
- 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. 黑龙江八一农垦大学生命科学技术学院,黑龙江大庆 163319;中国农业大学动物医学院,北京 100193
- 折叠
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rZHLV-U6-ZsGreen1-puro-MSTRG.22610.1和rZHLV-U6-ZsGreen1-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3 μg/mL,慢病毒感染的最适MOI为80.将rZHLV-U6-ZsGreen1-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-qPCR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-qPCR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-1 10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 mL、104.41 TCID50/0.1 mL及104.58 TCID50/0.1 mL.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1ncRNA-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促进病毒复制的分子机制及深入了解BHV-1感染的分子机制提供参考依据.
Abstract
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 rZHLV-U6-ZsGreen1-puro-MSTRG.22610.1 and rZHLV-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/mL,the optimal working concentration of puromycin was 3μg/mL,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 rZHLV-U6-ZsGreenl-puro-MSTRG.22610.1 and 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 and 1NC 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.22610.1 in 1T cell line was significantly higher than in 1NC and negative control cells(P＜0.0001).The results showed that a cell line with high expression of lncRNA-MSTRG.22610.1 without affecting cell proliferation was obtained,and the cell line was genetically stable.The 10-fold dilution of BHV-1 was used to infect the tenth passage of 1T and 1NC cells,respectively.The virus titer in each group was calculated using the Reed-Muench method 24 hours after infection.The results showed that the virus titers were 105.15 TCID50/0.1 mL,104.41 TCID50/0.1 mL,and 104.58 TCID50/0.1 mL for BHV-1 after infection of 1T,1NC,and MB cells(MDBK cells infected with BHV-1),respectively.Compared with MB and 1NC cells,the virus titer in 1T cells was significantly increased(P＜0.05),indicating the expression of lncRNA-MSTRG.22610.1 promoted the replication of BHV-1 in MDBK cells.This study provides a reference for further exploring the molecular mechanism of lncRNA-MSTRG.22610.1 promoting viral replication and understanding the molecular mechanism of BHV-1 infection.
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Key words
long non-coding RNA/MDBK cells/bovine herpesvirus-1/lentivirus overexpression/virus proliferation引用本文复制引用
基金项目
国家自然科学基金面上项目(32272991)
中国农业大学"小组团"援疆团队项目()
出版年
2024
NETL
NSTL
万方数据