二维类石墨烯材料抗病毒机理研究

发布时间：2018-01-22 09:59

本文关键词： 猪流行性腹泻病毒伪狂犬病毒氧化石墨烯抗病毒二维片状纳米材料　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：由细菌和病毒引起的传染病对人类及动物的健康造成了极大的危害,虽然有些传染病通过疫苗得到了控制,甚至根除,但大部分的传染病仍未得到很好的控制。不同血清型及毒株之间交叉保护率弱、新变异毒株不断出现等使得传染病的防控愈发艰难,因此,研究病毒致病机理及开发有效的抗病毒制剂对病毒性疾病的防控和治疗有着重要意义。本研究构建稳定表达PEDV ORF3的Vero细胞系,研究ORF3基因对细胞及PEDV病毒复制的影响,为揭示PEDV的致病机理提供理论依据。研究了二维片状纳米材料对PEDV及PRV的抗病毒作用及其可能的抗病毒机理,为新型抗病毒材料的研发提供基础。具体研究内容如下:1.构建稳定表达PEDV ORF3的Vero细胞系,分析ORF3对细胞周期、囊泡形成及病毒增殖的影响。通过慢病毒系统获得稳定表达PEDV ORF3的Vero细胞系,传代后仍能稳定表达ORF3。流式细胞术检测Vero及Vero-ORF3的细胞周期分布发现Vero-ORF3细胞中超过40%的细胞聚集在S期,而在Vero细胞为28.03%,表明表达ORF3可延长Vero细胞的S期。另一方面,超薄切片可以看出Vero-ORF3中囊泡的数量明显多于Vero细胞及转染截短ORF3的Vero细胞,并且还在Vero-ORF3细胞中观察到双层膜结构的囊泡,表明ORF3可促进细胞囊泡的形成,与病毒的复制密切相关。经TCID50及qPCR结果验证,两种PEDV弱毒株(AH-M及CV777疫苗株)在Vero-ORF3细胞上的量均高于Vero细胞,但强毒株(YN株)却没有表现出差异,Western blot结果也表明Vero-ORF3上PEDV N蛋白表达量明显高于Vero细胞。这些结果表明ORF3可促进PEDV弱毒株的增殖,但对强毒株没有影响。2.氧化石墨烯抗病毒研究通过噬斑形成试验、间接免疫荧光及Western blot验证,我们发现氧化石墨烯纳米材料对伪狂犬病毒(DNA病毒)及猪流行性腹泻病毒(RNA病毒)均有具有很好的抗病毒效果,并且这种抑制效果具有时间及剂量依赖效应。在病毒感染不同阶段加入GO发现其对病毒的抑制作用仅发生在病毒进入细胞之前,表明GO抗病毒作用发生在细胞外,很可能为直接接触发生的物理作用。为揭示了氧化石墨烯抗病毒机制,我们比较了GO及rGO抗病毒效果,发现它们效果一致,表明GO抗病毒作用不依赖于含氧基团。通过比较石墨、氧化石墨及GO发现多层片状的的氧化石墨抗病毒效果明显弱于单层结构的GO及rGO,而无定形的石墨不表现抗病毒作用,表明GO独特的单层结构对其抗病毒作用至关重要。随后,我们利用阳离子聚合物PDDA与GO复合,带正电荷的GO-PDDA复合物的抗病毒作用消失,而将GO与非离子聚合物PVP结合后仍带负电,保持其抗病毒活性,表明负电荷也在GO的抗病毒作用中有重要作用,因此我们推测GO可通过静电作用与病毒直接接触,利用其锋利的边缘结构破坏病毒结构,从而发挥抗病毒作用。3.类石墨烯材料MoS_2及WS_2抗病毒研究我们还发现其它二维片状纳米材料二硫化钼、二硫化钨也具有良好的抗病毒效果,但与氧化石墨烯相比,其抑制作用降低,并且其细胞毒性也比GO低。随后对MoS_2及GO抗病毒效果进行动物实验,评价其体内抑制病毒的能力。以猪伪狂犬病毒HNX株(103TCID50)感染Balb/c小鼠,攻毒组小鼠全部死亡,而GO及MoS_2与PRV孵育后攻毒组均有60%小鼠存活;攻毒后静脉注射GO组小鼠全部死亡,而注射MoS_2组小鼠还有40%小鼠存活。GO+PRV、MoS_2+PRV及MoS_2治疗组可对小鼠提供保护,并且小鼠发病时间延后1 d,且小鼠脑组织内病毒含量减少,表明MoS_2及GO可抑制PRV在体内的增殖,并且在感染后注射MoS_2仍具有保护效果。4.MoS_2及GO小鼠毒性研究为验证MoS_2及GO的毒性,采取动物攻毒实验中20倍治疗剂量(GO为2 mg/kg体重,MoS_2为10 mg/kg体重)静脉注射小鼠,分别于2、14、60 d取小鼠进行组织切片及血液学检测,结果表明小鼠主要实质器官(肺、肝、肾、脑)与对照组并无差异,未表现实质损伤,血常规及血清生化检测肝、肾功能各项指标也未见异常,证明我们实验浓度下MoS_2及GO对小鼠并无明显毒性作用。本研究发现PEDV ORF3可使细胞S期延长,并促进细胞囊泡的形成,从而促进PEDV弱毒的增殖,为PEDV致病机理研究提供基础。我们也首次发现了二维类石墨烯材料具有优越的抗病毒效果,并揭示了其中可能的机制,为新型抗病毒材料的研发提供依据。
[Abstract]:Health caused by bacterial and viral infectious disease to human and animal caused great harm, although some infectious diseases have been controlled by the vaccine, even eradicated, but most of the infectious disease has not been well controlled. Among different serotypes of strains and cross protection was weak, the new variant strains appear such as prevention and control infectious diseases more difficult, therefore, has important significance in prevention and treatment of viral pathogenesis and development of effective antiviral drugs for viral diseases. This study constructs Vero cell line with stable expression of PEDV ORF3, the research of ORF3 gene on the cell and the effect of PEDV virus replication, and provide a theoretical basis for the pathogenesis of PEDV the study of the antiviral effect. Two dimensional sheet of nano materials on PEDV and PRV and its antiviral mechanism may provide a basis for the development of new antiviral material. The concrete research Contents are as follows: 1. to construct a stable expression of PEDV ORF3 in Vero cell line, ORF3 analysis of the cell cycle, effect of vesicle formation and virus proliferation. Vero cells expressing PEDV ORF3 stability by lentiviral system. After passage, the expression is stable ORF3. cell cycle was detected by flow cytometry and Vero Vero-ORF3 distribution Vero-ORF3 more than 40% of the cells in the accumulation of cells in S phase in Vero cells was 28.03%, showed that the expression of ORF3 Vero cells can prolong the period of S. On the other hand, Vero-ORF3 can be seen in thin sections of the number of vesicles was higher than Vero cells and Vero cells transfected with truncated ORF3, and in Vero-ORF3 cells was observed in the double the membrane structure of vesicles, showed that ORF3 can promote the cell vesicle formation, and viral replication is closely related with TCID50 and qPCR. Results show that two kinds of PEDV (AH-M and CV777 attenuated vaccine strain) in Vero-ORF3 cells. The amount was higher than that of Vero cells, but the virulent strain (YN strain) did not show differences, Western blot results indicated that Vero-ORF3 PEDV N expression was significantly higher than that of Vero cells. These results indicate that ORF3 can promote the proliferation of PEDV strain, but the virulent strain.2. did not affect the oxidation of graphene by plaque antiviral research formation test, indirect immunofluorescence and Western blot verification, we found that graphene oxide nano materials of pseudorabies virus (DNA virus) and porcine epidemic diarrhea virus (RNA virus) has good antiviral effect, and the inhibitory effect was dose and time dependent. In different stages of accession to the GO virus infection found inhibition of virus only occurs before the virus enters a cell that GO antiviral effects occur in cells, physical action is likely to occur for direct contact. In order to reveal the graphite oxide Ene antiviral mechanism, we compared GO and rGO antiviral effect, they found the same effect that GO does not depend on the antiviral effect of oxygen containing groups. Through the comparison of graphite, graphite oxide and GO showed that GO and rGO graphite oxide multilayer chip the antiviral effect was weaker than that of monolayer structure, amorphous graphite does not exhibit antiviral effect showed that the unique structure of GO is crucial to its antiviral effect. Then, we use PDDA and GO cationic polymer composite, the antiviral effect of GO-PDDA complexes disappeared with a positive charge, while the GO and PVP combined with nonionic polymers still negatively charged, maintain its antiviral activity, showed that the negative charge also plays an important role in the antiviral effect of GO, so we speculate that GO can contact directly through the electrostatic interaction with the virus, the virus destroyed the structure of sharp edge structure, so as to exert antiviral Use the.3. class of graphene materials MoS_2 and WS_2 antiviral research we also found other two-dimensional nano sheet material MoS2, two tungsten sulfide also has good antiviral effect, but compared with the graphene oxide, reduce the inhibitory effect, and its toxicity is lower than that of GO. Then the animal experiments were carried out on MoS_2 and GO antiviral effect, ability to evaluate the in vivo inhibition of virus. The pseudorabies virus HNX strain (103TCID50) infection in Balb/c mice challenged mice all died, while GO and MoS_2 were incubated with PRV survival after vaccination group had 60% mice; after infection of intravenous injection of GO mice all died, and the injection of MoS_2 mice and 40% mice the survival of.GO+PRV, MoS_2+PRV and MoS_2 group can provide protection to the mice, and the onset time of mice by 1 D, and the virus content in the brain tissue of mice decreased, indicates that MoS_2 and GO can inhibit the proliferation of PRV in vivo, and After infection of MoS_2 injection has protective effect of.4.MoS_2 and GO mice in order to verify MoS_2 and GO toxicity, take animal poison attack experiment 20 times the therapeutic dose (GO 2 mg/kg MoS_2 weight, 10 mg/kg body weight) intravenous injection in mice, 2,14,60 D mice were detected by histological section and blood. The results showed that mice mainly parenchymatous organs (lung, liver, kidney, brain) and there is no difference between the control group and did not show substantial damage, blood routine and serum biochemical detection of liver and kidney function indexes is no exception, and we prove that GO MoS_2 experimental concentration has no obvious toxic effect on mice. The study found that PEDV ORF3 can make the cell the S period is prolonged, and promotes cell vesicle formation, thereby promoting PEDV attenuated the proliferation, provide the basis for the study of pathogenic mechanism of PEDV. We have also found superior antiviral effect of two-dimensional graphene material has, and exposing the The possible mechanisms are shown to provide a basis for the development of new antiviral materials.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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