马立克病病毒miR-M4-5p宿主靶基因cDNA文库的构建及鉴定

doi:10.11843/j.issn.0366-6964.2018.02.014

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (2): 348-359.doi: 10.11843/j.issn.0366-6964.2018.02.014

马立克病病毒miR-M4-5p宿主靶基因cDNA文库的构建及鉴定

薛正飞^1,2, 滕蔓², 李会珍², 马圣明^2,3, 宋利娜², 张雅², 罗俊^2,3, 张改平^1,2*

1. 河南农业大学, 生命科学学院, 郑州 450002;
2. 河南省农业科学院动物免疫学重点实验室, 农业部动物免疫学重点实验室, 河南省动物免疫学重点实验室, 郑州 450002;
3. 河南科技大学, 动物科技学院, 动物疫病与公共安全重点实验室, 洛阳 471003

收稿日期:2017-07-22 出版日期:2018-02-23 发布日期:2018-02-23
通讯作者: 张改平,E-mail:zhanggaiping2003@163.com
作者简介:薛正飞(1991-),男,河南商丘人,硕士生,主要从事病毒分子致病机制研究,Tel:0371-65756056,E-mail:13783676078@163.com
基金资助:
国家自然科学基金（U1604232；31602050）；国家重点研发计划（2016YFD0500800）

Construction and Identification of the cDNA Library of Host mRNA Targets Recognized by miR-M4-5p Encoded by Marek's Disease Virus

XUE Zheng-fei^1,2, TENG Man², LI Hui-zhen², MA Sheng-ming^2,3, SONG Li-na², ZHANG Ya², LUO Jun^2,3, ZHANG Gai-ping^1,2*

1. College of Life Science, Henan Agricultural University, Zhengzhou 450002, China;
2. Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
3. Key Laboratory of Animal Disease and Public Safety, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China

Received:2017-07-22 Online:2018-02-23 Published:2018-02-23

摘要/Abstract

摘要：

马立克病病毒（MDV）是少数感染自然宿主后可诱发淋巴瘤的疱疹病毒之一，是研究肿瘤发生及发展的理想动物模型。目前已报道MDV可编码数十种miRNA，其中MDV-1编码的miR-M4-5p被鉴定为宿主细胞miR-155的同源物，由于miR-155与人类多种肿瘤性疾病密切相关，这意味着miR-M4-5p可能在MDV的致瘤过程中扮演重要角色。本研究旨在构建miR-M4-5p的宿主靶基因文库并进行初步鉴定。提取鸡胚成纤维细胞（CEF）总RNA，反转录合成cDNA，用hybrid-PCR扩增候选靶基因片段连接到pMD19-T载体，转化大肠杆菌JM109感受态细胞后挑选单克隆进行PCR鉴定及测序，通过BLAST比对分析，共获得88个宿主候选靶基因序列。优先选择miRNA结合靶点位于mRNA 3'-UTR且与miR-M4-5p种子序列（2~7 nt）完全互补配对的29个候选靶基因，构建报告基因载体进行双荧光素酶报告试验，通过三轮双荧光素酶报告试验最终初步鉴定5个宿主靶基因：PRICKLE1、COLA、BCAT1、ANTXR1和TECPR1，为进一步揭示miR-M4-5p的分子调控机制奠定了重要基础。

Abstract:

Marek's disease virus (MDV) is one of the oncogenic herpes viruses that can induce tumors in their natural hosts, which is considered to be an excellent model for investigating the biology, genetics, and immunology of tumorigenesis. Recently, a lot of microRNAs (miRNAs) have been identified in MDV genomes and the miR-M4-5p encoded by MDV-1 has been identified as a viral analog of cellular miR-155. Since miR-155 is a host miRNA associated with several cancers, miR-M4-5p may play a critical role in MDV oncogenesis. The present work was performed to construct a cDNA library and to primarily screen the putative host mRNA targets for miR-M4-5p. All hybrid-PCR products amplified from CEF RNA were harvested and cloned into pMD19-T vectors, then transformed into E. coli JM109 to produce a pool. The clones were selected and sequenced, using the online basic local alignment search tool (BLAST) to analyze the target sequences. A total of 88 candidate mRNA genes were obtained, 29 of which contained the predicted binding miRNA sites in the 3'-UTRs, complementary to the seed sequence of miR-M4-5p. After three rounds of dual fluorescence reporter assays (DLRA), five host genes including PRICKLE1, COLA, BCAT1, ANTXR1 and TECPR1 were primarily identified as the biological targets for miR-M4-5p. Our work provides an important basis for further studies on the molecular regulatory mechanism mediated by miR-M4-5p.

中图分类号:

S852.659.1

薛正飞, 滕蔓, 李会珍, 马圣明, 宋利娜, 张雅, 罗俊, 张改平. 马立克病病毒miR-M4-5p宿主靶基因cDNA文库的构建及鉴定[J]. 畜牧兽医学报, 2018, 49(2): 348-359.

XUE Zheng-fei, TENG Man, LI Hui-zhen, MA Sheng-ming, SONG Li-na, ZHANG Ya, LUO Jun, ZHANG Gai-ping. Construction and Identification of the cDNA Library of Host mRNA Targets Recognized by miR-M4-5p Encoded by Marek's Disease Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 348-359.

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马立克病病毒miR-M4-5p宿主靶基因cDNA文库的构建及鉴定

Construction and Identification of the cDNA Library of Host mRNA Targets Recognized by miR-M4-5p Encoded by Marek's Disease Virus

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