BLV-miRNAs对牛乳过氧化物酶的影响
Effect of BLV-miRNAs on milk lactoperoxidase
王彦迪 1刘桂池 1杨跃杰 1王建发 1连帅 1武瑞1
作者信息
- 1. 黑龙江八一农垦大学动物科技学院农业农村部东北寒区牛病防治重点实验室/黑龙江省牛病防制重点实验室,黑龙江大庆 163319
- 折叠
摘要
为探究牛白血病病毒(bovine leukemia virus,BLV)编码的miRNAs(BLV-miRNAs)对奶牛乳腺上皮细胞(BMECs)乳过氧化物酶(LPO)的影响.首先通过ELISA测定高低不同载量BLV阳性奶牛牛乳中LPO的含量;随后体外培养BMECs,分别感染(1 MOI)BLV全长和缺失miRNAs的BLV(BLV-Δ miRNAs),通过荧光定量PCR、ELISA等方法检测各组BMECs LPO的表达水平;再通过StarMir等软件程序预测10种BLV-miRNAs是否靶向LPO;根据靶基因预测结果,进行BLV-miRNAs转染和靶基因初步验证.结果显示:BLV高载量阳性奶牛和低载量阳性奶牛牛乳中的LPO含量比阴性奶牛牛乳中的LPO分别降低了 8.73％和9.68％;体外试验结果显示:与感染BLV全长病毒组相比,缺失BLV-miRNAs后BMECs中的LPO表达水平显著升高(P＜0.05);靶位点预测结果显示BLV-miR-B1-5p、B3-3p和B4-5p可共同靶向LPO基因;转染BLV-miR-B1-5p到BMECs后LPO的基因表达显著下调(P＜0.05),并随着转染剂量的增多,LPO的表达水平呈下降趋势;转染BLV-miR-B4-3p到BMECs后LPO的基因表达显著下调(P＜0.05),但没有剂量依赖性.综上所述,BLV-miRNAs可以抑制BMECs中LPO的表达.
Abstract
This study aims to investigate the effect of bovine leukemia virus encoded microRNAs(BLV-miRNAs)on lactoperoxidase(LPO)in bovine mammary epithelial cells(BMECs).Firstly,the LPO content in the milk of BLV-positive cows,categorized by different viral loads,was quanti-fied by enzyme-linked immunosorbent assay(ELISA).Subsequently,BMECs were cultured in vitro and infected with(1 MOI)full-length BLV and BLV lacking miRNAs(BLV-ΔmiRNAs).Va-rious analytical techniques,including fluorescent quantitative PCR and ELISA,were used to assess LPO expression levels in different BMEC cohorts.Ten BLV-miRNAs were computationally predic-ted to target LPO using software tools such as StarMir.Based on these predictions,transfections of BLV-miRNAs were carried out and preliminary verification of their effects on target genes were performed.The results showed that compared to that of BLV-negative counterparts,the LPO levels in the milk of BLV-positive cows,stratified by high and low viral load,decreased by 8.73％and 9.68％,respectively.Invitro experiments further corroborated these trends,revealing a significant increase(P＜0.05)in LPO expression within BMECs following the deletion of BLV-miRNAs compared to the group infected with full-length BLV-infected group.Computational target site pre-dictions implicated BLV-miR-B1-5p,B3-3p,and B4-5p in the collective regulation of the LPO gene.Transfection of BLV-miR-B1-5p into BMECs resulted in a significant downregulation of LPO gene expression(P＜0.05),with the effect intensifying proportionally with the transfection dose.Simi-larly,the transfection of BLV-miR-B4-3p into BMECs significantly reduced LPO gene expression(P＜0.05)without showing a dose-dependent behavior.In conclusion,this research indicates that BLV-miRNAs can suppress LPO expression in BMECs.
关键词
牛白血病病毒/乳过氧化物酶/BLV-miRNAs/奶牛乳腺上皮细胞Key words
bovine leukemia virus/lactoperoxidase/BLV-miRNAs/bovine mammary epithelial cells引用本文复制引用
基金项目
国家自然科学基金(32002247)
国家自然科学基金(31972747)
黑龙江省自然科学基金(LH2023C083)
出版年
2024
NETL
NSTL
万方数据