VP1蛋白A289T变异影响肠道病毒71型感染神经系统的初步研究

发布时间：2018-05-15 03:33

本文选题：EV71病毒 + VP1蛋白　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景和目的肠道病毒71型(EV71)是手足口病的主要病原体,尤其是重症手足口病的主要病因(80%以上)。手足口病重症患者往往伴有EV71的神经系统感染,并且易留下神经系统后遗症,甚至出现死亡。目前,EV71甚至已经成为超越脊髓灰质炎病毒的最主要的中枢神经毒性的病毒,因此预防EV71的神经系统感染是降低手足口病死亡率的关键,也是手足口病防治的重点。相关研究发现EV71病毒的毒力改变与病毒基因组内部编码的氨基酸的突变有密切关系。也有文献证明VIM是细胞表面EV71受体。本课题组前期的流行病学研究发现:EV71病毒的结构蛋白VP1的A289T的变异与重症手足口病的发生密切关联,当289位点的氨基酸为A丙氨酸时,EV71感染神经系统明显升高(P0.05,OR=2.36,95%CI为1.163-4.659)。同时证实Vimentin是HBMEC细胞表面EV71的受体,并影响EV71在细胞中的复制与释放。本研究拟进一步对以上结果进行生物学实验验证。通过反向遗传学技术定点突变病毒VP1蛋白289位点、拯救病毒,利用体外实验和动物模型研究突变前后病毒的毒力及特性,初步探索VP1蛋白的A289T变异对EV71病毒感染神经系统的影响。利用体外实验和动物模型进一步的研究Vimentin影响EV71病毒感染。研究方法1.EV71病毒定点突变与病毒拯救运用分子克隆技术、反向遗传学技术将EV71-289A感染性质粒扩增后,定点突变为EV71-289T感染性质粒。野生型和突变型质粒经体外转录得到RNA,病毒RNA转染宿主细胞,获得两种拯救病毒。2.病毒生物特性的检测将两种拯救病毒感染RD细胞,运用细胞技术、PCR、Westemblot等技术,进行病毒的感染性、活性、稳定性、病毒定量的检测;以及两种病毒在HBMEC细胞中感染情况的比较。3.病毒感染动物模型建立动物模型,经腹腔注射分别感染两种拯救病毒,观察小鼠的病变情况,统计两种病毒的发病情况,并且用实时荧光定量PCR检测在小鼠的脑组织中进行病毒定量,HE染色观察脑组织的病变;用免疫组织化学方法检测脑组织中的病毒颗粒;进行两种病毒的毒力或者特性比较。结果1.EV71-289T感染性克隆突变成功;EV71A、EV71T病毒的拯救完成。2.两种拯救病毒在RD细胞中的侵袭、复制扩增、释放能及病毒自身蛋白合成能力相同,两者差异无统计学意义(P0.05)。3.两种拯救病毒可感染HBMEC细胞。相同MOI感染HBMEC细胞,EV71A病毒粘附量多于EV71T病毒(P0.05),EV71病毒在HBMEC对照细胞(CON)的吸附量是 VIM-KO 细胞的 1.2 倍多[CON:EV71A(1.00±0.15),EV71T(0.58±0.14),VIM-KO:EV71A(0.75±0.06),EV71T(0.47±0.20)]。MOI=10,病毒感染对照细胞24h、48h,细胞内EV71A核酸含量分别是EV71T核酸量的1.3 倍、3 倍[24h:EV71A(13.7±0.63),EV71T(10.62±0.64),48h:EV71A(100.42±0.85),EV71T(30.48±0.42)];VIM-KO 细胞中分别是 1.7 倍、1.8倍[24h:EV71A(2.94±0.96),EV71T(1.72±0.88),48h:EV71A(6.58±0.48),EV71T(3.58±0.81)](P0.05);细胞上清中EV71A核酸含量分别是EV71T的 2.2 倍、1.6 倍[24h:EV71A(1.00±0.05),EV71T(0.44±0.05),48h:EV71A(8.82±0.16),EV71T(5.35±0.07)];VIM-KO 细胞上清的分别是 1.4 倍、1.2倍[24h:EV71A(0.27±0.05),EV71T(0.18±0.08),48h:EV71A(0.68±0.06),EV71T(0.54±0.05)];VIM-KO中病毒核酸含量小于CON对照细胞(P0.05)。4.病毒感染BABL/c小鼠发病情况:108pfu病毒量:EV71A组发病率95.12%(39/41),EV71T 组发病率 60.47%(26/43);107pfu 病毒量:EV71A 组发病率36.36%(8/22),EV71T 组发病率 11.76%(2/17)(P0.05)。5.病毒感染SV129小鼠发病情况:VIM+/+SV129小鼠:108pfu病毒量:EV71A 组发病率 69.23%(27/39),EV71T 组发病率 42.5%(17/40)(P0.05);107pfu 病毒量:EV71A 组发病率 52.78%(19/36),EV71T 组发病率 22.86%(8/35)(P0.05);VIM-/-SV129小鼠:108pfu病毒量,两病毒组发病率均为10%(1/10);107pfu病毒量,两实验组均未发病。6.EV71A组小鼠脑组织病毒含量约为EV71T组的9.5倍[EV71A(2256.66±50.22),EV71T(237.75±36.15)](P0.05)。结论1.EV71病毒VP1蛋白A289T氨基酸变异后,病毒EV71毒力降低。2.验证了 VIM蛋白是HBMEC细胞的EV71受体,VIM蛋白是SV129小鼠体内EV71的受体。VIM影响EV71的粘附、复制以及病毒释放能力。
[Abstract]:Background and objective enterovirus 71 (EV71) is the main pathogen of hand foot and mouth disease (HFMD), especially the main cause of severe hand foot and mouth disease (more than 80%). Patients with HFMD are often associated with EV71 infection, and are prone to sequelae of the nervous system and even death. At present, EV71 has even become a transcendental gray matter of the spinal cord. The most important central neurotoxic virus of the virus, so the prevention of EV71 infection is the key to reducing the mortality of hand foot and mouth disease, and is also the key to the prevention and treatment of hand foot and mouth disease. Related studies have found that the virulence of the EV71 virus is closely related to the mutation of the amino acids encoded in the genome of the virus. It is also documented that the VIM is the same. EV71 receptor on the surface of the cell. The previous epidemiological study found that the variation of the A289T of the structural protein VP1 of the EV71 virus was closely related to the occurrence of severe hand foot and mouth disease. When the amino acid at the 289 site was A alanine, the nervous system of EV71 infection was significantly elevated (P0.05, OR= 2.36,95%CI was 1.163-4.659). Meanwhile, Vimentin was confirmed as HBMEC thin. The receptor of EV71 on the cell surface affects the replication and release of EV71 in the cell. This study intends to further verify the results of the above results by biological experiments. Through the reverse genetics technique, the 289 site of the site directed mutagenesis virus VP1 protein is used to save the virus and to explore the virulence and characteristics of the virus before and after the mutation by using the in vitro experiment and animal model. The preliminary exploration of the VP1 The effect of A289T variation on EV71 virus infection in the nervous system. Using in vitro experiments and animal models to further study the effect of Vimentin on the infection of EV71 virus. Research methods 1.EV71 virus fixed-point mutation and virus rescue use molecular cloning technology. After amplification of EV71-289A infectious plasmids by reverse genetics technology, the fixed-point mutation is EV71-28 9T infection particles. Wild type and mutant plasmid are transcribed in vitro to RNA, virus RNA is transfected into host cells. The detection of two kinds of rescue virus.2. virus biological characteristics will infect two RD cells, using cell technology, PCR, Westemblot and other techniques to detect virus infection, activity, stability, and quantitative detection of virus; And comparing the infection of two viruses in HBMEC cells, the animal model of.3. virus infected animal model was established. Two kinds of rescue viruses were infected by intraperitoneal injection. The pathological changes of the mice were observed and the incidence of the two viruses was observed. The quantitative detection of the virus in the brain tissues of the mice was carried out by real-time fluorescence quantitative PCR detection, and the HE staining concept was used. Detect the pathological changes of the brain tissue; detect the virus particles in the brain tissue by immunohistochemistry; compare the virulence or characteristics of the two viruses. Results the 1.EV71-289T cloned mutation was successful; the rescue of EV71A and EV71T virus completed the invasion of the two kinds of virus in RD cells, the replication and amplification, the release energy and the virus self protein combination. The difference was not significant (P0.05), and there was no statistically significant difference (P0.05).3. two kinds of rescue viruses can infect HBMEC cells. The same MOI infection HBMEC cells, EV71A virus adhesion more than EV71T virus (P0.05), EV71 virus in HBMEC control cells (CON) is more than 1.2 times more than VIM-KO cells (1 + 0.15), 0.58 + 0.14 (0.58 + 0.14). 0.75 + 0.06), EV71T (0.47 + 0.20)].MOI=10, the virus infected control cells 24h, 48h, and the content of EV71A nucleic acid in the cell is 1.3 times of EV71T nucleic acid, 3 times [24h:EV71A (13.7 + 0.63), EV71T (10.62 + 0.64), 48h:EV71A (100.42 + 0.85), EV71T (30.48 +)]. 8), 48h:EV71A (6.58 + 0.48), EV71T (3.58 + 0.81)] (P0.05); the content of EV71A nucleic acid in the cell supernatant is 2.2 times of EV71T, 1.6 times [24h:EV71A (1 + 0.05), EV71T (0.44 + 2.2), 48h:EV71A (8.82 + 0.16), EV71T (8.82). (+ 0.06), EV71T (0.54 + 0.05)]; the content of viral nucleic acid in VIM-KO was less than that of CON control cells (P0.05).4. virus infected BABL/c mice: the amount of 108pfu virus: the incidence of EV71A group was 95.12% (39/41), the incidence rate of EV71T group was 60.47% (26/43), the incidence of 107pfu virus was 36.36%, and the incidence of 11.76% group was 11.76%. Infection of SV129 mice: VIM+/+SV129 mice: 108pfu virus: the incidence of 108pfu virus: the incidence of EV71A group was 69.23% (27/39), the incidence of EV71T group was 42.5% (17/40) (P0.05), 107pfu virus was 52.78% (19/36), and the incidence of EV71T group was 22.86%, and the incidence of two virus group was 10%; 107 The amount of PFU virus in the two experimental group did not occur in the.6.EV71A group. The virus content in the brain tissue of the.6.EV71A group was approximately [EV71A (2256.66 + 50.22) and EV71T (237.75 + 36.15)] (P0.05). Conclusion the EV71 virulence of the 1.EV71 virus VP1 protein was variable, and the EV71 virulence of the virus EV71 was.2.. The receptor.VIM of EV71 affects the adhesion, replication and virus release ability of EV71.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.5

【参考文献】