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首页> 外文期刊>Fish & Shellfish Immunology >Generation of microRNA-30e-producing recombinant viral hemorrhagic septicemia virus (VHSV) and its effect on in vitro immune responses
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Generation of microRNA-30e-producing recombinant viral hemorrhagic septicemia virus (VHSV) and its effect on in vitro immune responses

机译：产生MicroRNA-30E产生的重组病毒出血性败血症（VHSV）及其对体外免疫应答的影响

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摘要

MicroRNAs (miRNAs) are non-coding small RNAs involved in the regulation of gene expression. In the present study, we firstly reported the use of a fish RNA virus, viral hemorrhagic septicemia virus (VHSV), as a delivery vehicle of a miRNA-30e, and the effect of miR-30e produced by the recombinant VHSV on the immune responses of Epithelioma papulosum cyprini (EPC) cells was investigated. The expression of functional miR-30e using a CMV promoter-driven vector was verified by the significantly lower eGFP expression in cells transfected with a vector containing miR-30e sponge sequence than that in cells transfected with a control vector that had mutated miR-30e sponge sequence. Furthermore, the down-regulation of reporter gene containing 3'-UTR of NF-kappa b inhibitor alpha-like protein B (NF kappa bi alpha b) by miR-30e was demonstrated, suggesting that miR-30e overexpression can increase immune responses related to NF-kappa B activation through inhibition of I kappa B. A miR-30e-expressing recombinant VHSV (rVHSV-A-miR30e) that had primary microRNA-30e sequence between N and P genes was rescued using the reverse genetic method, and the successful expression of miR-30e in the cells infected with rVHSV-A-miR30e was demonstrated using Northern blot and qRT-PCR. Cells infected with rVHSV-A-miR30e showed the increase of NF-kappa B activation and type I interferon induced genes expression, suggesting that rVHSVA-miR30e can produce functional miR-30e in fish cells, and VHSV can be used as a vehicle to deliver functional microRNAs in fish.
机译：MicroRNA（miRNA）是非编码的小RNA，参与基因表达的调节。在本研究中，我们首先报道了使用鱼RNA病毒，病毒出血性杂草病毒（VHSV）作为miRNA-30e的递送载体，以及通过重组VHSV产生的miR-30e对免疫应答产生的影响研究了上皮瘤Cyprini（EPC）细胞进行了研究。使用CMV启动子驱动载体的功能性miR-30e的表达通过用含有miR-30e海绵序列的载体转染的细胞中的显着降低的EGFP表达来验证，比用突变的MIR-30E海绵转染的细胞中的细胞中的细胞序列。此外，对MiR-30E进行了含有NF-Kappa抑制剂α样NF-κB抑制剂α样蛋白B（NF Kappa Bi alpha B）的报告基因的下调，表明miR-30e过表达可以增加相关的免疫应答通过抑制I Kappa B的NF-Kappa B激活。使用反向遗传方法拯救在N和P基因之间具有初级microRNA-30E序列的miR-30E表达重组VHSV（RVHSV-A-MIR30E），使用Northern印迹和QRT-PCR证明了使用RVHSV-A-miR30e感染的细胞中miR-30e的成功表达。感染RVHSV-A-miR30E的细胞显示NF-κB激活和I型干扰素诱导基因表达的增加，表明RVHSVA-MIR30E可以在鱼细胞中产生功能性miR-30e，并且VHSV可以用作送达的车辆鱼类的功能性micrornas。

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