抗猪繁殖与呼吸综合征病毒药物的高通量筛选方法的建立
发布时间：2018-11-23 07:50
【摘要】：猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是一种以妊娠母猪繁殖障碍和生长仔猪呼吸道症状为主要临床特征的传染病,其病原体为猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)。PRRSV属于动脉炎病毒科(Arterivirus),为不分节段的单股正链RNA病毒。该病毒具有高度的变异性,易形成异源的种群,目前没有有效的疫苗和药物进行防治。本研究利用PRRSV非结构蛋白加工成熟时的特异性切割序列,构建萤火虫荧光素酶系统指示PRRSV的增殖水平,并通过多种抑制剂和药物进行验证,拟为抗PRRSV药物的筛选建立一种新型的药物高通量筛选方法。具体研究内容如下:1、确定最佳的荧光素酶报告系统本研究将PRRSV 12个非结构蛋白的切割序列分别与萤火虫荧光素酶融合表达,构建了12个荧光素酶报告质粒,建立PRRSV感染增殖指示生物传感器。将报告质粒分别转染Marc-145细胞,在PRRSV感染细胞的过程中,病毒的蛋白酶会切割这些特异性的序列,从而激活该荧光素酶报告系统。根据荧光素酶报告系统激活的程度,筛选到最佳的荧光素酶报告系统为358-DnaE-Nsp3/4。2、荧光素酶报告系统的特异性和敏感性分析为了验证358-DnaE-Nsp3/4荧光素酶报告系统的特异性和敏感性,本研究将PRRSV编码的蛋白酶Nsp4和358D-Nsp3/4报告质粒在HEK-293T细胞中共表达,发现Nsp4能切割该报告质粒中的特异性序列,激活荧光素酶报告系统。将Nsp4蛋白酶活性中心的3个氨基酸进行特异性突变,使其切割活性丧失,将突变体与358D-Nsp3/4报告质粒在HEK-293T细胞中共表达,发现突变体不能激活荧光素酶报告系统,表明该荧光素酶报告系统是特异性依赖PRRSV编码的蛋白酶Nsp4的活性。通过PRRSV、PRV和TGEV感染实验,发现仅PRRSV感染能激活该荧光素酶报告系统,表明该系统能特异性的指示PRRSV的感染和增殖。此外,PRRSV感染激活该荧光素酶报告系统的能力具有剂量依赖性,表明该系统还能对PRRSV的增殖情况进行定量分析。3、荧光素酶报告系统在抗PRRSV药物筛选中的应用将358D-Nsp3/4荧光素酶报告系统与NSP4共转染到HEK-293T细胞后,加入4种不同的药物,分别是EPDTC、RID、AMA与RUP。发现只有EPDTC与RUP能抑制荧光素酶的活性,表明这2种蛋白酶抑制剂能抑制该报告系统的活性。将358D-Nsp3/4荧光素酶报告质粒转染到Marc-145细胞中,PRRSV感染后分别加入不同的药物,同时,以噬斑减数试验检测药物处理后PRRSV的增殖滴度。发现该荧光素酶活性的变化能真实反应病毒滴度的变化。这些结果表明该荧光素酶报告系统能够作为一种新型抗PRRSV药物的高通量筛选方法,具有一定的应用前景。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28
本文编号：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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28
【参考文献】
相关期刊论文 前1条
1 ;Caspase Family Proteases and Apoptosis[J];Acta Biochimica et Biophysica Sinica;2005年11期
相关博士学位论文 前1条
1 王荡;口蹄疫病毒L~（pro）和3C~（pro）调控宿主抗病毒天然免疫反应的分子机制研究[D];华中农业大学;2011年
，本文编号：2350829
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2350829.html
