调控HO-1表达的转录因子的鉴定及其在PRRSV感染中的作用研究
发布时间：2019-06-02 08:16
【摘要】：猪繁殖与呼吸综合征(PRRS)在世界范围内广泛存在,并且从经济学角度来讲,是养猪生产实践中最重要的传染性疾病之一。1987年最早在美国被报导,几年后荷兰也发现了PRRS。这种疾病表现出很多临床症状而生产母猪、后备母猪严重的繁殖障碍(主要表现为怀孕后期流产、木乃伊胎、死胎和弱胎的增多)和所有日龄阶段猪呼吸道问题是两种最主要的症状。由于PRRSV抗原具备的诸多特点如抗原的高突变性,ADE(抗体依赖性增强作用)还有持续性感染等使得PRRS的防治难上加难,而且目前对该病和抗原的研究始终没有突破性的进展。然而,近几年对细胞血红素加氧酶(HO-1)的抗炎、抗氧化应激和抗病毒作用的研究日益增多,本项目组最近研究发现HO-1的激动剂CoPP诱导表达和腺病毒介导的HO-1过表达均可显著抑制PRRSV感染Marc-145细胞和猪肺泡巨噬细胞(PAM),表明HO-1具有抗PRRSV感染的作用。本研究拟从转录水平研究HO-1抗PRRSV感染的作用和分子机制,鉴定出对HO-1基因表达起调控作用的关键转录因子,为确立HO-1作为PRRS防治新靶点提供充分科学依据。研究结果:1.HO-1全长启动子的克隆、鉴定与初步分析利用Promoter2.0 Prediction等多种软件预测HO-1启动子区序列并设计引物结合Infusion技术获得含有启动子区长度为6553bp的荧光素酶报告载体,通过测序得到了启动子区全长序列,并通过荧光素酶活性检测确定其具有启动子活性。2.HO-1核心启动子区域的筛选通过构建一系列顺序截断启动子区荧光素酶报告载体,结合荧光素报告基因活性检测结果,将HO-1的基础启动子区定位在(-440~-121)之间,只要含有此片段就能够启动HO-1的转录;将HO-1增强型启动子区确定在(-2065~-2021)44bp区间,在含有基础启动子的条件下此片段的荧光素酶活性是基础启动子区的7倍;还发现了上游存在转录负调控区(-6553~-6316)238bp,此片段存在时可使启动子区活性降低40%。3.关键转录因子的预测分析和Nrf2的鉴定综合利用软件预测获得与调控HO-1转录相关一些转录因子,结合启动子定点突变和荧光素酶双报告基因检测证实HO-1基因启动子核心区的Nrf2结合位点确实能够调控HO-1的转录,Nrf2结合位点突变后此片段的荧光素酶活性丢失。4.转录因子Nrf2在HO-1调控PRRSV感染过程中的作用初探利用Nrf2的特异性诱导剂tBHQ对其进行诱导表达,qPCR检测Nrf2和HO-1的mRNA水平,Nrf2和HO-1随着tBHQ浓度和时间的变化呈正相关的关系。选取tBHQ的最佳作用时间和浓度对Marc145细胞进行处理,然后接毒检测Nrf2、HO-1和N基因mRNA的表达量,初步确定tBHQ通过诱导Nrf2进而调控HO-1抵抗PRRSV的感染。综上所述,转录因子Nrf2能够通过调控HO-1转录进而抵抗PRRSV感染,这为PRRSV的防治提供了新的思路。
[Abstract]:Pig reproductive and respiratory syndrome (PRRS) is widely found in the world, and from an economic point of view, it is one of the most important infectious diseases in pig production. It was first reported in the United States in 1987, and PRRS. was also discovered in the Netherlands a few years later. The disease shows many clinical symptoms and gives birth to sows, and reserve sows have serious reproductive disorders (mainly due to abortion in the second trimester of pregnancy, mummies. The increase in stillbirths and weak births) and respiratory problems in pigs at all ages are the two main symptoms. Because of many characteristics of PRRSV antigen, such as highly mutagenicity, ADE (antibody dependent enhancement of antigen) and persistent infection, the prevention and treatment of PRRS is even more difficult, and there has been no breakthrough in the study of PRRS antigen and antigen. However, in recent years, there have been more and more studies on the anti-inflammatory, anti-oxidative stress and antiviral effects of heme oxygenase (HO-1). Recent studies in our project team found that the expression induced by CoPP, an agonist of HO-1, and the overexpression of HO-1 mediated by adenoviruses could significantly inhibit the infection of Marc-145 cells by PRRSV and the (PAM), of alveolar macrophages in pigs. HO-1 had the effect of anti-PRRSV infection. The purpose of this study was to study the anti-PRRSV infection effect and molecular mechanism of HO-1 at transcriptional level, and to identify the key transcription factors that regulate the expression of HO-1 gene, so as to provide sufficient scientific basis for the establishment of HO-1 as a new target for PRRS prevention and treatment. Results: the full-length promoter of 1.HO-1 was cloned. Identification and preliminary analysis of HO-1 promoter region sequence predicted by Promoter2.0 Prediction and other software, primers were designed and Infusion technique was designed to obtain luciferase report vector containing promoter region length 6553bp, and the full length sequence of promoter region was obtained by sequencing. The promoter activity was determined by luciferase activity detection. 2.The core promoter region of Ho-1 was screened by constructing a series of luciferase report vectors with sequential truncation of promoter region, combined with the results of luciferin reporter gene activity detection. The basic promoter region of HO-1 is located between (- 1940 鈮，
本文编号：2490952
[Abstract]:Pig reproductive and respiratory syndrome (PRRS) is widely found in the world, and from an economic point of view, it is one of the most important infectious diseases in pig production. It was first reported in the United States in 1987, and PRRS. was also discovered in the Netherlands a few years later. The disease shows many clinical symptoms and gives birth to sows, and reserve sows have serious reproductive disorders (mainly due to abortion in the second trimester of pregnancy, mummies. The increase in stillbirths and weak births) and respiratory problems in pigs at all ages are the two main symptoms. Because of many characteristics of PRRSV antigen, such as highly mutagenicity, ADE (antibody dependent enhancement of antigen) and persistent infection, the prevention and treatment of PRRS is even more difficult, and there has been no breakthrough in the study of PRRS antigen and antigen. However, in recent years, there have been more and more studies on the anti-inflammatory, anti-oxidative stress and antiviral effects of heme oxygenase (HO-1). Recent studies in our project team found that the expression induced by CoPP, an agonist of HO-1, and the overexpression of HO-1 mediated by adenoviruses could significantly inhibit the infection of Marc-145 cells by PRRSV and the (PAM), of alveolar macrophages in pigs. HO-1 had the effect of anti-PRRSV infection. The purpose of this study was to study the anti-PRRSV infection effect and molecular mechanism of HO-1 at transcriptional level, and to identify the key transcription factors that regulate the expression of HO-1 gene, so as to provide sufficient scientific basis for the establishment of HO-1 as a new target for PRRS prevention and treatment. Results: the full-length promoter of 1.HO-1 was cloned. Identification and preliminary analysis of HO-1 promoter region sequence predicted by Promoter2.0 Prediction and other software, primers were designed and Infusion technique was designed to obtain luciferase report vector containing promoter region length 6553bp, and the full length sequence of promoter region was obtained by sequencing. The promoter activity was determined by luciferase activity detection. 2.The core promoter region of Ho-1 was screened by constructing a series of luciferase report vectors with sequential truncation of promoter region, combined with the results of luciferin reporter gene activity detection. The basic promoter region of HO-1 is located between (- 1940 鈮，
本文编号：2490952
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