調(diào)控HO-1表達(dá)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(shí)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(jì)學(xué)角度來(lái)講,是養(yǎng)豬生產(chǎn)實(shí)踐中最重要的傳染性疾病之一。1987年最早在美國(guó)被報(bào)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(diǎn)如抗原的高突變性,ADE(抗體依賴性增強(qiáng)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(duì)該病和抗原的研究始終沒有突破性的進(jìn)展。然而,近幾年對(duì)細(xì)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(xiàng)目組最近研究發(fā)現(xiàn)HO-1的激動(dòng)劑CoPP誘導(dǎo)表達(dá)和腺病毒介導(dǎo)的HO-1過(guò)表達(dá)均可顯著抑制PRRSV感染Marc-145細(xì)胞和豬肺泡巨噬細(xì)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(jī)制,鑒定出對(duì)HO-1基因表達(dá)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(diǎn)提供充分科學(xué)依據(jù)。研究結(jié)果:1.HO-1全長(zhǎng)啟動(dòng)子的克隆、鑒定與初步分析利用Promoter2.0 Prediction等多種軟件預(yù)測(cè)HO-1啟動(dòng)子區(qū)序列并設(shè)計(jì)引物結(jié)合Infusion技術(shù)獲得含有啟動(dòng)子區(qū)長(zhǎng)度為6553bp的熒光素酶報(bào)告載體,通過(guò)測(cè)序得到了啟動(dòng)子區(qū)全長(zhǎng)序列,并通過(guò)熒光素酶活性檢測(cè)確定其具有啟動(dòng)子活性。2.HO-1核心啟動(dòng)子區(qū)域的篩選通過(guò)構(gòu)建一系列順序截?cái)鄦?dòng)子區(qū)熒光素酶報(bào)告載體,結(jié)合熒光素報(bào)告基因活性檢測(cè)結(jié)果,將HO-1的基礎(chǔ)啟動(dòng)子區(qū)定位在(-440~-121)之間,只要含有此片段就能夠啟動(dòng)HO-1的轉(zhuǎn)錄;將HO-1增強(qiáng)型啟動(dòng)子區(qū)確定在(-2065~-2021)44bp區(qū)間,在含有基礎(chǔ)啟動(dòng)子的條件下此片段的熒光素酶活性是基礎(chǔ)啟動(dòng)子區(qū)的7倍;還發(fā)現(xiàn)了上游存在轉(zhuǎn)錄負(fù)調(diào)控區(qū)(-6553~-6316)238bp,此片段存在時(shí)可使啟動(dòng)子區(qū)活性降低40%。3.關(guān)鍵轉(zhuǎn)錄因子的預(yù)測(cè)分析和Nrf2的鑒定綜合利用軟件預(yù)測(cè)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(dòng)子定點(diǎn)突變和熒光素酶雙報(bào)告基因檢測(cè)證實(shí)HO-1基因啟動(dòng)子核心區(qū)的Nrf2結(jié)合位點(diǎn)確實(shí)能夠調(diào)控HO-1的轉(zhuǎn)錄,Nrf2結(jié)合位點(diǎn)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(guò)程中的作用初探利用Nrf2的特異性誘導(dǎo)劑tBHQ對(duì)其進(jìn)行誘導(dǎo)表達(dá),qPCR檢測(cè)Nrf2和HO-1的mRNA水平,Nrf2和HO-1隨著tBHQ濃度和時(shí)間的變化呈正相關(guān)的關(guān)系。選取tBHQ的最佳作用時(shí)間和濃度對(duì)Marc145細(xì)胞進(jìn)行處理,然后接毒檢測(cè)Nrf2、HO-1和N基因mRNA的表達(dá)量,初步確定tBHQ通過(guò)誘導(dǎo)Nrf2進(jìn)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(guò)調(diào)控HO-1轉(zhuǎn)錄進(jìn)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(hào)：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(hào)：2490952
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