抗豬繁殖與呼吸綜合征病毒藥物的高通量篩選方法的建立
發(fā)布時(shí)間：2018-11-23 07:50
【摘要】：豬繁殖與呼吸綜合征(porcine reproductive and respiratory syndrome,PRRS)是一種以妊娠母豬繁殖障礙和生長仔豬呼吸道癥狀為主要臨床特征的傳染病,其病原體為豬繁殖與呼吸綜合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)。PRRSV屬于動脈炎病毒科(Arterivirus),為不分節(jié)段的單股正鏈RNA病毒。該病毒具有高度的變異性,易形成異源的種群,目前沒有有效的疫苗和藥物進(jìn)行防治。本研究利用PRRSV非結(jié)構(gòu)蛋白加工成熟時(shí)的特異性切割序列,構(gòu)建螢火蟲熒光素酶系統(tǒng)指示PRRSV的增殖水平,并通過多種抑制劑和藥物進(jìn)行驗(yàn)證,擬為抗PRRSV藥物的篩選建立一種新型的藥物高通量篩選方法。具體研究內(nèi)容如下:1、確定最佳的熒光素酶報(bào)告系統(tǒng)本研究將PRRSV 12個(gè)非結(jié)構(gòu)蛋白的切割序列分別與螢火蟲熒光素酶融合表達(dá),構(gòu)建了12個(gè)熒光素酶報(bào)告質(zhì)粒,建立PRRSV感染增殖指示生物傳感器。將報(bào)告質(zhì)粒分別轉(zhuǎn)染Marc-145細(xì)胞,在PRRSV感染細(xì)胞的過程中,病毒的蛋白酶會切割這些特異性的序列,從而激活該熒光素酶報(bào)告系統(tǒng)。根據(jù)熒光素酶報(bào)告系統(tǒng)激活的程度,篩選到最佳的熒光素酶報(bào)告系統(tǒng)為358-DnaE-Nsp3/4。2、熒光素酶報(bào)告系統(tǒng)的特異性和敏感性分析為了驗(yàn)證358-DnaE-Nsp3/4熒光素酶報(bào)告系統(tǒng)的特異性和敏感性,本研究將PRRSV編碼的蛋白酶Nsp4和358D-Nsp3/4報(bào)告質(zhì)粒在HEK-293T細(xì)胞中共表達(dá),發(fā)現(xiàn)Nsp4能切割該報(bào)告質(zhì)粒中的特異性序列,激活熒光素酶報(bào)告系統(tǒng)。將Nsp4蛋白酶活性中心的3個(gè)氨基酸進(jìn)行特異性突變,使其切割活性喪失,將突變體與358D-Nsp3/4報(bào)告質(zhì)粒在HEK-293T細(xì)胞中共表達(dá),發(fā)現(xiàn)突變體不能激活熒光素酶報(bào)告系統(tǒng),表明該熒光素酶報(bào)告系統(tǒng)是特異性依賴PRRSV編碼的蛋白酶Nsp4的活性。通過PRRSV、PRV和TGEV感染實(shí)驗(yàn),發(fā)現(xiàn)僅PRRSV感染能激活該熒光素酶報(bào)告系統(tǒng),表明該系統(tǒng)能特異性的指示PRRSV的感染和增殖。此外,PRRSV感染激活該熒光素酶報(bào)告系統(tǒng)的能力具有劑量依賴性,表明該系統(tǒng)還能對PRRSV的增殖情況進(jìn)行定量分析。3、熒光素酶報(bào)告系統(tǒng)在抗PRRSV藥物篩選中的應(yīng)用將358D-Nsp3/4熒光素酶報(bào)告系統(tǒng)與NSP4共轉(zhuǎn)染到HEK-293T細(xì)胞后,加入4種不同的藥物,分別是EPDTC、RID、AMA與RUP。發(fā)現(xiàn)只有EPDTC與RUP能抑制熒光素酶的活性,表明這2種蛋白酶抑制劑能抑制該報(bào)告系統(tǒng)的活性。將358D-Nsp3/4熒光素酶報(bào)告質(zhì)粒轉(zhuǎn)染到Marc-145細(xì)胞中,PRRSV感染后分別加入不同的藥物,同時(shí),以噬斑減數(shù)試驗(yàn)檢測藥物處理后PRRSV的增殖滴度。發(fā)現(xiàn)該熒光素酶活性的變化能真實(shí)反應(yīng)病毒滴度的變化。這些結(jié)果表明該熒光素酶報(bào)告系統(tǒng)能夠作為一種新型抗PRRSV藥物的高通量篩選方法,具有一定的應(yīng)用前景。
[Abstract]:Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome,PRRS) is an infectious disease characterized by reproductive disorders in pregnant sows and respiratory symptoms in growing piglets. The pathogen is porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus,). PRRSV). PRRSV belongs to the single strand positive strand RNA virus (Arterivirus),) of the arteritis virus family, which is not segmented. The virus has high variability, easy to form heterologous populations, there are no effective vaccines and drugs to prevent and cure. In this study, the specific cleavage sequence of PRRSV nonstructural protein was used to construct a fluorescent luciferase system to indicate the level of PRRSV proliferation, which was verified by a variety of inhibitors and drugs. To establish a new high-throughput screening method for anti-PRRSV drugs. The main contents of this study are as follows: 1. The optimal luciferase reporting system was established. In this study, 12 luciferase reporter plasmids were constructed by fusion of the cleavage sequence of 12 nonstructural proteins of PRRSV with luciferase of firefly. To establish PRRSV infection indicator biosensor. The reporter plasmids were transfected into Marc-145 cells respectively. In the process of PRRSV infection, the protease of the virus cleans these specific sequences and activates the luciferase reporting system. According to the degree of activation of luciferase reporting system, the best luciferase reporting system was 358-DnaE-Nsp3 / 4.2. Specificity and sensitivity Analysis of luciferase reporting system in order to verify the specificity and sensitivity of 358-DnaE-Nsp3/4 luciferase reporting system, In this study, the protease Nsp4 and 358D-Nsp3/4 reporter plasmids encoded by PRRSV were co-expressed in HEK-293T cells. It was found that Nsp4 could cut the specific sequence of the reporter plasmid and activate the luciferase reporting system. Three amino acids in the active center of Nsp4 protease were specifically mutated and the cleavage activity was lost. The mutants and 358D-Nsp3/4 reporter plasmids were co-expressed in HEK-293T cells. It was found that the mutants could not activate the luciferase reporting system. The results showed that the luciferase report system was specific to the activity of protease Nsp4 encoded by PRRSV. PRRSV,PRV and TGEV infection experiments showed that only PRRSV infection could activate the luciferase reporting system, indicating that the system could specifically indicate the infection and proliferation of PRRSV. In addition, the ability of PRRSV infection to activate the luciferase report system is dose-dependent, indicating that the system can also quantitatively analyze the proliferation of PRRSV. Application of luciferase reporting system in screening of anti- PRRSV drugs the 358D-Nsp3/4 luciferase report system was cotransfected with NSP4 into HEK-293T cells and four different drugs were added, namely EPDTC,RID,AMA and RUP.. It was found that only EPDTC and RUP could inhibit luciferase activity, indicating that these two protease inhibitors could inhibit the activity of the report system. 358D-Nsp3/4 luciferase reporter plasmid was transfected into Marc-145 cells. Different drugs were added to Marc-145 cells after PRRSV infection. The proliferative titer of PRRSV after drug treatment was detected by plaque reduction test. It was found that the change of luciferase activity could reflect the change of virus titer. These results indicate that the luciferase report system can be used as a new high-throughput screening method for anti-PRRSV drugs and has a certain application prospect.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S858.28
本文編號：2350829
[Abstract]:Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome,PRRS) is an infectious disease characterized by reproductive disorders in pregnant sows and respiratory symptoms in growing piglets. The pathogen is porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus,). PRRSV). PRRSV belongs to the single strand positive strand RNA virus (Arterivirus),) of the arteritis virus family, which is not segmented. The virus has high variability, easy to form heterologous populations, there are no effective vaccines and drugs to prevent and cure. In this study, the specific cleavage sequence of PRRSV nonstructural protein was used to construct a fluorescent luciferase system to indicate the level of PRRSV proliferation, which was verified by a variety of inhibitors and drugs. To establish a new high-throughput screening method for anti-PRRSV drugs. The main contents of this study are as follows: 1. The optimal luciferase reporting system was established. In this study, 12 luciferase reporter plasmids were constructed by fusion of the cleavage sequence of 12 nonstructural proteins of PRRSV with luciferase of firefly. To establish PRRSV infection indicator biosensor. The reporter plasmids were transfected into Marc-145 cells respectively. In the process of PRRSV infection, the protease of the virus cleans these specific sequences and activates the luciferase reporting system. According to the degree of activation of luciferase reporting system, the best luciferase reporting system was 358-DnaE-Nsp3 / 4.2. Specificity and sensitivity Analysis of luciferase reporting system in order to verify the specificity and sensitivity of 358-DnaE-Nsp3/4 luciferase reporting system, In this study, the protease Nsp4 and 358D-Nsp3/4 reporter plasmids encoded by PRRSV were co-expressed in HEK-293T cells. It was found that Nsp4 could cut the specific sequence of the reporter plasmid and activate the luciferase reporting system. Three amino acids in the active center of Nsp4 protease were specifically mutated and the cleavage activity was lost. The mutants and 358D-Nsp3/4 reporter plasmids were co-expressed in HEK-293T cells. It was found that the mutants could not activate the luciferase reporting system. The results showed that the luciferase report system was specific to the activity of protease Nsp4 encoded by PRRSV. PRRSV,PRV and TGEV infection experiments showed that only PRRSV infection could activate the luciferase reporting system, indicating that the system could specifically indicate the infection and proliferation of PRRSV. In addition, the ability of PRRSV infection to activate the luciferase report system is dose-dependent, indicating that the system can also quantitatively analyze the proliferation of PRRSV. Application of luciferase reporting system in screening of anti- PRRSV drugs the 358D-Nsp3/4 luciferase report system was cotransfected with NSP4 into HEK-293T cells and four different drugs were added, namely EPDTC,RID,AMA and RUP.. It was found that only EPDTC and RUP could inhibit luciferase activity, indicating that these two protease inhibitors could inhibit the activity of the report system. 358D-Nsp3/4 luciferase reporter plasmid was transfected into Marc-145 cells. Different drugs were added to Marc-145 cells after PRRSV infection. The proliferative titer of PRRSV after drug treatment was detected by plaque reduction test. It was found that the change of luciferase activity could reflect the change of virus titer. These results indicate that the luciferase report system can be used as a new high-throughput screening method for anti-PRRSV drugs and has a certain application prospect.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S858.28
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