發(fā)布時(shí)間：2018-05-15 03:33

  本文選題：EV71病毒 + VP1蛋白�。� 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景和目的腸道病毒71型(EV71)是手足口病的主要病原體,尤其是重癥手足口病的主要病因(80%以上)。手足口病重癥患者往往伴有EV71的神經(jīng)系統(tǒng)感染,并且易留下神經(jīng)系統(tǒng)后遺癥,甚至出現(xiàn)死亡。目前,EV71甚至已經(jīng)成為超越脊髓灰質(zhì)炎病毒的最主要的中樞神經(jīng)毒性的病毒,因此預(yù)防EV71的神經(jīng)系統(tǒng)感染是降低手足口病死亡率的關(guān)鍵,也是手足口病防治的重點(diǎn)。相關(guān)研究發(fā)現(xiàn)EV71病毒的毒力改變與病毒基因組內(nèi)部編碼的氨基酸的突變有密切關(guān)系。也有文獻(xiàn)證明VIM是細(xì)胞表面EV71受體。本課題組前期的流行病學(xué)研究發(fā)現(xiàn):EV71病毒的結(jié)構(gòu)蛋白VP1的A289T的變異與重癥手足口病的發(fā)生密切關(guān)聯(lián),當(dāng)289位點(diǎn)的氨基酸為A丙氨酸時(shí),EV71感染神經(jīng)系統(tǒng)明顯升高(P0.05,OR=2.36,95%CI為1.163-4.659)。同時(shí)證實(shí)Vimentin是HBMEC細(xì)胞表面EV71的受體,并影響EV71在細(xì)胞中的復(fù)制與釋放。本研究擬進(jìn)一步對以上結(jié)果進(jìn)行生物學(xué)實(shí)驗(yàn)驗(yàn)證。通過反向遺傳學(xué)技術(shù)定點(diǎn)突變病毒VP1蛋白289位點(diǎn)、拯救病毒,利用體外實(shí)驗(yàn)和動(dòng)物模型研究突變前后病毒的毒力及特性,初步探索VP1蛋白的A289T變異對EV71病毒感染神經(jīng)系統(tǒng)的影響。利用體外實(shí)驗(yàn)和動(dòng)物模型進(jìn)一步的研究Vimentin影響EV71病毒感染。研究方法1.EV71病毒定點(diǎn)突變與病毒拯救運(yùn)用分子克隆技術(shù)、反向遺傳學(xué)技術(shù)將EV71-289A感染性質(zhì)粒擴(kuò)增后,定點(diǎn)突變?yōu)镋V71-289T感染性質(zhì)粒。野生型和突變型質(zhì)粒經(jīng)體外轉(zhuǎn)錄得到RNA,病毒RNA轉(zhuǎn)染宿主細(xì)胞,獲得兩種拯救病毒。2.病毒生物特性的檢測將兩種拯救病毒感染RD細(xì)胞,運(yùn)用細(xì)胞技術(shù)、PCR、Westemblot等技術(shù),進(jìn)行病毒的感染性、活性、穩(wěn)定性、病毒定量的檢測;以及兩種病毒在HBMEC細(xì)胞中感染情況的比較。3.病毒感染動(dòng)物模型建立動(dòng)物模型,經(jīng)腹腔注射分別感染兩種拯救病毒,觀察小鼠的病變情況,統(tǒng)計(jì)兩種病毒的發(fā)病情況,并且用實(shí)時(shí)熒光定量PCR檢測在小鼠的腦組織中進(jìn)行病毒定量,HE染色觀察腦組織的病變;用免疫組織化學(xué)方法檢測腦組織中的病毒顆粒;進(jìn)行兩種病毒的毒力或者特性比較。結(jié)果1.EV71-289T感染性克隆突變成功;EV71A、EV71T病毒的拯救完成。2.兩種拯救病毒在RD細(xì)胞中的侵襲、復(fù)制擴(kuò)增、釋放能及病毒自身蛋白合成能力相同,兩者差異無統(tǒng)計(jì)學(xué)意義(P0.05)。3.兩種拯救病毒可感染HBMEC細(xì)胞。相同MOI感染HBMEC細(xì)胞,EV71A病毒粘附量多于EV71T病毒(P0.05),EV71病毒在HBMEC對照細(xì)胞(CON)的吸附量是 VIM-KO 細(xì)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,病毒感染對照細(xì)胞24h、48h,細(xì)胞內(nèi)EV71A核酸含量分別是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 細(xì)胞中分別是 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);細(xì)胞上清中EV71A核酸含量分別是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 細(xì)胞上清的分別是 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對照細(xì)胞(P0.05)。4.病毒感染BABL/c小鼠發(fā)病情況:108pfu病毒量:EV71A組發(fā)病率95.12%(39/41),EV71T 組發(fā)病率 60.47%(26/43);107pfu 病毒量:EV71A 組發(fā)病率36.36%(8/22),EV71T 組發(fā)病率 11.76%(2/17)(P0.05)。5.病毒感染SV129小鼠發(fā)病情況:VIM+/+SV129小鼠:108pfu病毒量:EV71A 組發(fā)病率 69.23%(27/39),EV71T 組發(fā)病率 42.5%(17/40)(P0.05);107pfu 病毒量:EV71A 組發(fā)病率 52.78%(19/36),EV71T 組發(fā)病率 22.86%(8/35)(P0.05);VIM-/-SV129小鼠:108pfu病毒量,兩病毒組發(fā)病率均為10%(1/10);107pfu病毒量,兩實(shí)驗(yàn)組均未發(fā)病。6.EV71A組小鼠腦組織病毒含量約為EV71T組的9.5倍[EV71A(2256.66±50.22),EV71T(237.75±36.15)](P0.05)。結(jié)論1.EV71病毒VP1蛋白A289T氨基酸變異后,病毒EV71毒力降低。2.驗(yàn)證了 VIM蛋白是HBMEC細(xì)胞的EV71受體,VIM蛋白是SV129小鼠體內(nèi)EV71的受體。VIM影響EV71的粘附、復(fù)制以及病毒釋放能力。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.5

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本文編號：1890806