猪繁殖与呼吸障碍综合征病毒NSP10 DNA结合活性的研究及抑制剂的高通量筛选

发布时间：2018-03-19 16:29

本文选题：猪繁殖与呼吸障碍综合征病毒(PRRSV)　切入点：nsp10　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸障碍综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)属于动脉炎病毒科动脉炎病毒属成员,基因组为单股正链RNA,大小约为15kb,含有9个开放阅读框。主要引起妊娠母猪繁殖障碍和仔猪呼吸道疾病。猪繁殖与呼吸障碍综合征是一种病毒性传染病,已在全世界流行,给养猪业带来严重损失。目前为止,该病没有特异性治疗方法且疫苗防治效果不理想。只有进一步研究病毒复制机理及其相关蛋白功能,寻找针对PRRSV特异性预防与治疗策略,对于控制该病发生,减少养猪业的经济损失具有重要意义。PRRSV nsp10蛋白为一种多功能蛋白,由N端的锌结合结构域(Zinc-binding domain,ZBD)和C端的超家族1解螺旋酶域组成,具有结合活性和解旋酶活性。ZBD由一个a-螺旋和两个反平行的β-折叠共三个肽段组成,a-螺旋能镶嵌于DNA的大沟中,从而结合DNA或者RNA。本实验主要对PRRSV nsp10蛋白解旋活性与结合活性进行了研究,在细胞水平上进行抗病毒药物的筛选,为该病毒的致病机理研究及药物预防与治疗提供参考。主要工作如下:1.Nsp10蛋白解旋方向及其底物突出端最小长度的检测利用非变性聚丙烯酰胺凝胶实验方法验证nsp10的解旋活性,测定蛋白的解旋方向以及底物突出端所需的最小长度。实验证明nsp10具有5’端到3’端解旋活性,底物5’突出端的长度应大于5bp,才能保证解旋反应的正常进行。2.Nsp10蛋白的ss DNA及ds DNA结合活性检测Nsp10蛋白N-端存在锌结合结构域,可以与DNA相结合,因此利用凝胶阻滞实验检测了蛋白核酸结合活性,结果表明,nsp10具有非特异性的ss DNA与ds DNA结合活性,且不需要ATP的参与。3.锌结合结构域突变体蛋白的表达纯化及活性检测通过对动脉炎病毒科动脉炎病毒属四种(EAV、LDV、SHFV、PRRSV)病毒ZBD区域的对比,以及根据EAV晶体结构的分析,选择6个氨基酸进行突变,结果显示C10、C25、H28、H32可溶性表达发生变化,尝试不同表达载体p ET-28a-SUMO、p GEX6p-1进行摸索,以获得可溶性表达。突变体蛋白在相同浓度下,检测其解旋活性以及结合活性,C25、H32位活性消失,为ZBD的关键位点。4.化合物库的高通量筛选在细胞水平上建立了抗PRRSV高通量筛选方法,最终确定Marc-145细胞最适的接板密度5000 cells/孔、检测时间120h以及0.01个MOIs病毒的感染剂量。并且在满足S/B5、Z’≥0.5和CV10%条件下对实验室的LOPAC1280库进行筛选,得到了抑制率大于60%的6种化合物,分析复筛结果,考虑到临床实际应用,选择其中两种A1895、D8555进行后续实验。5.抗PRRSV化合物的检测A1895、D8555具有价格便宜,抑制率高的优点,通过Western blot和间接免疫荧光实验检测两种化合物的抑制效果,结果显示,两种药物的抗病毒效果良好。A1895为DNA拓扑异构酶ⅱ抑制剂,D8555为高亲和力配体神经胶质线粒体苯二氮受体,是一种抗焦虑药。
[Abstract]:Porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) belongs to the Arteriviridae arterivirus member genome is a single strand RNA, about 15KB in size, containing 9 open reading frames. The main cause reproductive failure in pregnant sows and piglets respiratory diseases. Porcine reproductive and respiratory disorders in general syndrome is a viral infectious disease, which has been popular in the world, caused serious losses to the pig industry. So far, the disease has no specific treatment and vaccine prevention effect is not ideal. Only further research on the function mechanism of virus replication and related protein, in order to find PRRSV specific prevention and treatment strategies for the control of the disease occurrence, reduce economic losses in pig industry has important significance of.PRRSV Nsp10 protein is a multifunctional protein, from the N side of the zinc binding domain (Zinc-binding domain, ZB D) and C terminal superfamily 1 helicase domain, the binding activity and helicase activity of.ZBD consists of a a- helix and two antiparallel beta sheet total of three peptides, a- can be embedded in the major groove of spiral DNA, which combined with DNA or RNA. in the experiment of PRRSV Nsp10 protein Jiexuan activity and binding activity were studied. The screening of antiviral drugs at the cellular level, and provide a reference for the study of pathogenic mechanism and drug prevention and treatment of the virus. The main work is as follows: the activity of 1.Nsp10 protein solution solution spin spin direction and the minimum length of substrate protruding end detection using non denaturing polyacrylamide Nsp10 verification the gel test method, determination of the minimum length of protein unwinding direction and substrate protruding end required. Experiments show that Nsp10 is the 5 'end to the 3' end of helicase activity, substrate 5 'overhang length should be greater than 5bp, in order to ensure Unwinding reaction of normal SS DNA and DS DNA protein.2.Nsp10 binding activity of Nsp10 protein was detected at N- zinc binding domain, can be combined with DNA, so using gel retardation assay of protein nucleic acid binding activity, the results show that the binding activity of Nsp10 with nonspecific SS DNA and DS DNA, and do not need to ATP.3. in the zinc binding domain mutant expression purification and activity detection of proteins by Arteriviridae arteriviruses genera and four species (EAV, LDV, SHFV, PRRSV) of viral ZBD region, and according to the analysis of the crystal structure of EAV, 6 amino acid mutations, the results showed that C10, C25, H28. The expression changes of soluble H32, try a different expression vector of P ET-28a-SUMO and P GEX6p-1 were explored to obtain soluble expression of mutant protein. At the same concentration, detect the unwinding activity and binding activity, C25, H32 An activity disappeared for high-throughput key sites of.4. ZBD compound library screening at the cellular level established for anti PRRSV high-throughput screening methods, and ultimately determine the optimal Marc-145 cell plate density 5000 cells/ hole infection dose detection time 120h and 0.01 MOIs virus. And in the LOPAC1280 library to meet S/B5. Laboratory Z '= 0.5 and CV10% under the condition of 6 kinds of compounds were screened, the inhibition rate was higher than 60% were obtained. The analysis of screening results, considering the clinical application, select one of two kinds of A1895, A1895 D8555 detection for subsequent experiments of.5. anti PRRSV compounds, D8555 has the advantages of low price, restrain the advantage of high efficiency. The inhibitory effect of Western, blot and indirect immunofluorescence assay showed that two compounds, two kinds of drugs for good antiviral effect of DNA topoisomerase inhibitors.A1895, D8555 for high affinity ligands The neuroglia mitochondrial two nitrogen receptor is an anti anxiety drug.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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