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膜联蛋白A2是一种参与调节多种病毒增殖的重要宿主蛋白。本研究通过构建过表达膜联蛋白A2的BHKAnxa2细胞系及瞬时敲低膜联蛋白A2,探讨膜联蛋白A2对脑心肌炎病毒在BHK-21细胞中增殖的影响。通过RT-PCR从BHK-21细胞中扩增膜联蛋白A2全长cDNA,测序正确后克隆入pcDNA3.1整合表达载体中;用重组载体pcDNA3.1-Anxa2转染BHK-21细胞,经G418筛选获得抗性克隆,qRT-PCR和Western blot试验证明BHKAnxa2过表达膜联蛋白A2;EMCV感染试验证明BHK-Anxa2细胞中病毒滴度高于对照组。通过siRNA降低BHK-21细胞中膜联蛋白A2表达,EMCV感染试验证明膜联蛋白A2表达下降后EMCV增殖也随之下降。以上试验结果表明鼠膜联蛋白A2表达改变可导致病毒在BHK-21细胞中的增殖发生变化,提示膜联蛋白A2与EMCV在BHK-21细胞中的增殖相关。
Abstract：Annexin A2 is an important host factor that regulates several key processes in diverse viruses.To study the potential involvement of annexin A2 upon replication of the encephalomyocarditis virus(EMCV)in BHK-21 cells,overexpression and RNA interference(RNAi)were utilized.The full-length complementary DNA(cDNA)was amplified from BHK-21 cells by reverse transcription-polymerase chain reaction(RT-PCR),and subcloned into the eukaryotic expression vector pcDNA3.1.The recombinant vector pcDNA3.1-Anxa2 was transfected into BHK-21 cells and a transgenic cell line generated after G418 selection.Overexpression of annexin A2 in BHK-Anxa2 was detected by qRT-PCR and western blotting.EMCV challenge experiments demonstrated that the virus titer in BHK-Anxa2 was higher than that in the control group.Moreover,knockdown of annexin A2 using an RNAi approach inhibited EMCV infection in BHK-21 cells.These results suggest that the level of annexin A2 might participate in EMCV replication.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.003154
中图分类号:R373
引用信息:
[1]李琼毅,包晓婧,张海霞,等.膜联蛋白A2对脑心肌炎病毒在BHK-21细胞中增殖的影响[J].病毒学报,2017,33(03):331-337.DOI:10.13242/j.cnki.bingduxuebao.003154.
基金信息:
国家自然科学基金(项目号:31460665),题目:脑心肌炎病毒受体的筛选、鉴定及功能分析; 教育部“长江学者和创新团队发展计划”(项目号:IRT13091); 中央高校基本科研业务费专项资金(项目号:31920150025),题目:膜联蛋白 A2调节脑心肌炎病毒感染的作用和机制研究。
2017-05-25
2017-05-25
2017-05-25