調(diào)控HO-1表達的轉(zhuǎn)錄因子的鑒定及其在PRRSV感染中的作用研究
發(fā)布時間：2019-06-02 08:16
【摘要】：豬繁殖與呼吸綜合征(PRRS)在世界范圍內(nèi)廣泛存在,并且從經(jīng)濟學(xué)角度來講,是養(yǎng)豬生產(chǎn)實踐中最重要的傳染性疾病之一。1987年最早在美國被報導(dǎo),幾年后荷蘭也發(fā)現(xiàn)了PRRS。這種疾病表現(xiàn)出很多臨床癥狀而生產(chǎn)母豬、后備母豬嚴(yán)重的繁殖障礙(主要表現(xiàn)為懷孕后期流產(chǎn)、木乃伊胎、死胎和弱胎的增多)和所有日齡階段豬呼吸道問題是兩種最主要的癥狀。由于PRRSV抗原具備的諸多特點如抗原的高突變性,ADE(抗體依賴性增強作用)還有持續(xù)性感染等使得PRRS的防治難上加難,而且目前對該病和抗原的研究始終沒有突破性的進展。然而,近幾年對細胞血紅素加氧酶(HO-1)的抗炎、抗氧化應(yīng)激和抗病毒作用的研究日益增多,本項目組最近研究發(fā)現(xiàn)HO-1的激動劑CoPP誘導(dǎo)表達和腺病毒介導(dǎo)的HO-1過表達均可顯著抑制PRRSV感染Marc-145細胞和豬肺泡巨噬細胞(PAM),表明HO-1具有抗PRRSV感染的作用。本研究擬從轉(zhuǎn)錄水平研究HO-1抗PRRSV感染的作用和分子機制,鑒定出對HO-1基因表達起調(diào)控作用的關(guān)鍵轉(zhuǎn)錄因子,為確立HO-1作為PRRS防治新靶點提供充分科學(xué)依據(jù)。研究結(jié)果:1.HO-1全長啟動子的克隆、鑒定與初步分析利用Promoter2.0 Prediction等多種軟件預(yù)測HO-1啟動子區(qū)序列并設(shè)計引物結(jié)合Infusion技術(shù)獲得含有啟動子區(qū)長度為6553bp的熒光素酶報告載體,通過測序得到了啟動子區(qū)全長序列,并通過熒光素酶活性檢測確定其具有啟動子活性。2.HO-1核心啟動子區(qū)域的篩選通過構(gòu)建一系列順序截斷啟動子區(qū)熒光素酶報告載體,結(jié)合熒光素報告基因活性檢測結(jié)果,將HO-1的基礎(chǔ)啟動子區(qū)定位在(-440~-121)之間,只要含有此片段就能夠啟動HO-1的轉(zhuǎn)錄;將HO-1增強型啟動子區(qū)確定在(-2065~-2021)44bp區(qū)間,在含有基礎(chǔ)啟動子的條件下此片段的熒光素酶活性是基礎(chǔ)啟動子區(qū)的7倍;還發(fā)現(xiàn)了上游存在轉(zhuǎn)錄負調(diào)控區(qū)(-6553~-6316)238bp,此片段存在時可使啟動子區(qū)活性降低40%。3.關(guān)鍵轉(zhuǎn)錄因子的預(yù)測分析和Nrf2的鑒定綜合利用軟件預(yù)測獲得與調(diào)控HO-1轉(zhuǎn)錄相關(guān)一些轉(zhuǎn)錄因子,結(jié)合啟動子定點突變和熒光素酶雙報告基因檢測證實HO-1基因啟動子核心區(qū)的Nrf2結(jié)合位點確實能夠調(diào)控HO-1的轉(zhuǎn)錄,Nrf2結(jié)合位點突變后此片段的熒光素酶活性丟失。4.轉(zhuǎn)錄因子Nrf2在HO-1調(diào)控PRRSV感染過程中的作用初探利用Nrf2的特異性誘導(dǎo)劑tBHQ對其進行誘導(dǎo)表達,qPCR檢測Nrf2和HO-1的mRNA水平,Nrf2和HO-1隨著tBHQ濃度和時間的變化呈正相關(guān)的關(guān)系。選取tBHQ的最佳作用時間和濃度對Marc145細胞進行處理,然后接毒檢測Nrf2、HO-1和N基因mRNA的表達量,初步確定tBHQ通過誘導(dǎo)Nrf2進而調(diào)控HO-1抵抗PRRSV的感染。綜上所述,轉(zhuǎn)錄因子Nrf2能夠通過調(diào)控HO-1轉(zhuǎn)錄進而抵抗PRRSV感染,這為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，
本文編號：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，
本文編號：2490952
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