本文關(guān)鍵詞： 腸道 病毒 分離 檢測(cè) 活疫苗 免疫 原性 研究　出處：《昆明理工大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：手足口病(hand, foot and mouth disease, HFMD)是由腸道病毒引發(fā)的一種疾病,主要在5歲以下兒童中流行,傳染性強(qiáng),可引發(fā)多種中樞神經(jīng)系統(tǒng)并發(fā)癥,導(dǎo)致患兒死亡。目前,引發(fā)手足口病的病毒主要為柯薩奇病毒A組16型(CoxA16)(?)口腸道病毒71型(EV71)。由于手足口病發(fā)病迅速、在發(fā)病初期,臨床表現(xiàn)不明顯,難以在發(fā)病的初期進(jìn)行及時(shí)的診斷,目前針對(duì)手足口病的治療只限于發(fā)病后的對(duì)癥治療,沒有有效的抗病毒藥物,也沒有用于預(yù)防該病的疫苗。隨著近幾年由EV71病毒引發(fā)的手足口病在我國(guó)的大規(guī)模爆發(fā),我國(guó)很可能已經(jīng)成為了該病潛在爆發(fā)區(qū)[3,4,5,61, EV71是引起手足口病的重要病原微生物,特別是重癥感染、致死性感染的主要病因。在滅活疫苗、減毒活疫苗、亞單位疫苗、DNA疫苗、病毒樣顆粒疫苗、轉(zhuǎn)基因動(dòng)物疫苗中,滅活疫苗因具備較為完備的抗原表位和較為簡(jiǎn)單的制備條件,最具開發(fā)潛力的疫苗形式,然而EV71的特點(diǎn)之一就是具有高度的變異性,分離得到最新流行的病毒是制備滅活疫苗的基礎(chǔ)。 本課題的第一部分主要包括處理糞便、分離病毒；RT-PCR分群鑒定毒種；EV71噬斑形成實(shí)驗(yàn)可行性預(yù)實(shí)驗(yàn)；顯微挑斑法純化病毒；RT-PCR鑒定是否分離成功；病變形態(tài)學(xué)篩選EV71；VP1基因的菌落PCR和酶切鑒定；VP1基因全序測(cè)定及進(jìn)化樹的構(gòu)建；全序測(cè)定的菌落PCR鑒定、酶切鑒定；全序測(cè)定；EV71電鏡觀察；EV71免疫熒光實(shí)驗(yàn)。第二部分主要包括EV71MOI檢測(cè)；無血清維持培養(yǎng)Vero NaHCO3含量值條件摸索；EV71在細(xì)胞和上清中的分布；無血清維持培養(yǎng)EV71可行性研究,Vero細(xì)胞有／無血清維持培養(yǎng)EV71上清中病毒一步動(dòng)力曲線檢測(cè)；Vero細(xì)胞無血清維持培養(yǎng)EV71上清和細(xì)胞中病毒增殖動(dòng)力曲線；氯仿抽提、超聲對(duì)EV71滴度的影響；EV71傳代穩(wěn)定性研究。第三部分主要包括細(xì)胞、病毒增殖曲線,pH變化情況；純化、滅活病毒；EV71滅活疫苗免疫后總抗體的檢測(cè)；EV71滅活疫苗中和抗體檢測(cè)。 本文創(chuàng)新性改進(jìn)了病毒分離方法,明顯提高了病毒分離純化效率,通過這種方法成功的將EV71和CA16分離開來。經(jīng)過分型鑒定證明分離到的EV71為C4a亞型,并成功獲得全長(zhǎng)序列和電鏡照片。進(jìn)一步檢測(cè)了MOI、無血清培養(yǎng)pH條件、病毒在細(xì)胞內(nèi)外的增殖曲線等生物學(xué)特性。最后,將EV71滅活后免疫動(dòng)物,檢測(cè)到了較高滴度的免疫原性和中和抗體效價(jià)。
[Abstract]:Hand, foot and mouth disease (HFMD) is a disease caused by enterovirus, mainly in children under 5 years of age. Infectious, can cause a variety of central nervous system complications, leading to child death. At present, the main cause of hand, foot and mouth disease is Coxsackie virus group A CoxA16? Due to the rapid onset of hand, foot and mouth disease, the clinical manifestation is not obvious at the beginning of the disease, so it is difficult to make timely diagnosis in the early stage of the disease. At present, the treatment of hand, foot and mouth disease is limited to the symptomatic treatment after onset, there is no effective antiviral drugs. There is no vaccine to prevent the disease. With the massive outbreak of HFMD caused by EV71 virus in China in recent years, China is likely to have become a potential outbreak of the disease. [3,4,5,61, EV71 is an important pathogen of hand, foot and mouth disease (HFMD), especially the main cause of severe infection and fatal infection, including inactivated vaccine, live attenuated vaccine and subunit vaccine. Virus-like granular vaccine, transgenic animal vaccine, inactivated vaccine because of relatively complete antigen epitopes and relatively simple preparation conditions, the most potential form of vaccine development. However, one of the characteristics of EV71 is its high variability, the isolation of the latest prevalent virus is the basis for the preparation of inactivated vaccines. The first part of this subject mainly includes the disposal of feces and isolation of viruses. RT-PCR group identification of virus species; EV71 plaque formation experiment feasibility pretest; The virus was purified by microblotting method. RT-PCR was used to identify whether the separation was successful or not. EV71 was screened by pathological morphology. Colony PCR and enzyme digestion of VP1 gene; Total sequence determination of VP1 gene and construction of phylogenetic tree; Total sequence determination of colony PCR identification, enzyme digestion identification; Total sequence determination; EV71 electron microscopy; EV71 immunofluorescence assay. The second part mainly includes the detection of EV71MOI; The conditions of Vero NaHCO3 content in serum-free culture were explored. The distribution of EV71 in cells and supernatants; Feasibility study of serum-free maintenance culture of EV71 virus in supernatant of EV71 cultured with or without serum-free was detected by one step dynamic curve. Vero cells maintained the EV71 supernatant and cell proliferation dynamics curve without serum. The effect of ultrasound on the titer of EV71 by chloroform extraction; Study on the stability of EV71 passage. The third part mainly includes cell, virus proliferation curve and pH change; Purification, inactivation of virus; Detection of total antibody after immunization with inactivated EV71 vaccine; Detection of neutralizing antibody against EV71 inactivated vaccine. In this paper, the virus isolation method was innovatively improved, and the efficiency of virus isolation and purification was improved obviously. EV71 and CA16 were successfully separated by this method. The isolated EV71 was identified as C4a subtype. The full length sequence and electron microscope photos were obtained successfully. The biological characteristics of moi, the pH condition of serum-free culture, the proliferation curve of the virus in and out of cells were further detected. Finally, the EV71 was inactivated to immunize the animals. High titer immunogenicity and neutralizing antibody titer were detected.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1;R725.1

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