本文選題：腸道病毒71型 + 脊髓灰質(zhì)炎病毒��； 參考：《華東師范大學(xué)》2012年碩士論文

【摘要】：近年來(lái),手足口病(HFMD)在我國(guó)呈持續(xù)性流行態(tài)勢(shì),而且死亡病例逐年增加。腸道病毒71型(EV71)感染所引起的HFMD患者易導(dǎo)致嚴(yán)重的并發(fā)癥,如無(wú)菌性腦膜炎、腦干腦炎和脊髓灰質(zhì)炎樣的麻痹等神經(jīng)系統(tǒng)相關(guān)疾病,還可能引起更嚴(yán)重的肺水腫,伴有肺水腫的重癥病例病情進(jìn)展快,死亡率高。目前,EV71致病的分子機(jī)制尚不清楚,而且沒(méi)有針對(duì)這種病毒的特異性藥物及疫苗。本研究從體液免疫角度探索EV71抗體反應(yīng)的有關(guān)規(guī)律,以期為EV71流行病的致病機(jī)理、治療及疫苗研發(fā)提供一些理論參考。本項(xiàng)研究有兩方面的內(nèi)容：其一,通過(guò)臨床血清樣本研究及實(shí)驗(yàn)動(dòng)物水平的驗(yàn)證,探討EV71與脊髓灰質(zhì)炎病毒(PV)之間的抗體交叉反應(yīng)性；其二,通過(guò)酶聯(lián)免疫吸附試驗(yàn)ELISA實(shí)驗(yàn)及中和抑制實(shí)驗(yàn)研究SP70和SP55在EV71感染的人體內(nèi)能否誘導(dǎo)產(chǎn)生相應(yīng)的中和抗體。 ELISA檢測(cè)EV71或PV與健康兒童血清IgG的反應(yīng)性,研究發(fā)現(xiàn)EV71與PV陽(yáng)性EV71陰性健康兒童血清IgG有很高的反應(yīng)性(陽(yáng)性率達(dá)77.8%),而且在這些PV陽(yáng)性EV71陰性兒童血清中針對(duì)這兩種病毒的IgG在含量上呈正相關(guān)(r=0.58)。微量中和試驗(yàn)研究發(fā)現(xiàn),這些PV陽(yáng)性兒童血清不能中和EV71。EV71與PV陽(yáng)性但EV71陰性人血清孵育后感染人單核白血病細(xì)胞系(THP-1),感染后培養(yǎng)24小時(shí),以實(shí)時(shí)定量PCR檢測(cè)進(jìn)入THP-1的EV71病毒數(shù)量,結(jié)果發(fā)現(xiàn)PV陽(yáng)性EV71陰性的兒童血清能增強(qiáng)EV71感染進(jìn)入THP-1細(xì)胞。以上結(jié)果初步表明,PV疫苗接種能誘導(dǎo)產(chǎn)生與EV71交叉反應(yīng)性的抗體,雖然這些交叉反應(yīng)性抗體是非中和抗體,但這些非中和抗體在體外能促進(jìn)EV71感染THP-1,這表明PV接種產(chǎn)生的與EV71交叉反應(yīng)性抗體可能與EV71的致病有關(guān),所以本研究可能為EV71致病機(jī)制的研究開(kāi)辟一個(gè)可能的新方向。 為了在背景清晰的實(shí)驗(yàn)動(dòng)物水平進(jìn)一步驗(yàn)證EV71與PV之間的抗體交叉反應(yīng)性,以原核表達(dá)與親和純化等方法分別獲得EV71和PV的衣殼蛋白VP1及非結(jié)構(gòu)蛋白3C,將PV-VP1、EV71-VP1、EV71滅活疫苗及PV滅活疫苗(IPV)分別經(jīng)皮下免疫BALB/c小鼠制得免疫血清。蛋白免疫印跡實(shí)驗(yàn)顯示PV-VP1能與EV71-VP1抗血清反應(yīng),反之,EV71-VP1也能與PV-VP1抗血清反應(yīng)；競(jìng)爭(zhēng)ELISA試驗(yàn)進(jìn)一步顯示EV71-VP1蛋白能明顯抑制PV-VP1與其特異性免疫血清抗體的結(jié)合,反之亦然；但微量中和實(shí)驗(yàn)揭示PV陽(yáng)性小鼠血清不能中和EV71。SPF級(jí)小鼠實(shí)驗(yàn)研究證實(shí)EV71與PV之間確實(shí)存在大量交叉反應(yīng)性非中和抗體。 前人以小鼠實(shí)驗(yàn)研究發(fā)現(xiàn)兩個(gè)EV71的重要線性中和表位(SP70與SP55)。為了研究這兩個(gè)表位在人體內(nèi)的反應(yīng)性,合成這兩個(gè)表位,收集EV71感染兒童恢復(fù)期血清及未曾感染EV71的健康兒童血清,以ELISA檢測(cè)SP70或SP55與EV71感染患者恢復(fù)期血清IgG的反應(yīng)性。研究發(fā)現(xiàn),SP70或SP55與EV71感染者血清IgG的反應(yīng)性和與未曾感染的健康兒童血清IgG的反應(yīng)性沒(méi)有差異。EV71陽(yáng)性兒童血清分別與SP70或SP55預(yù)孵育后在體外中和EV71,研究發(fā)現(xiàn)這兩個(gè)肽不能抑制EV71陽(yáng)性人血清的中和效應(yīng)。本研究表明,在自然感染時(shí),SP70和SP55在人體內(nèi)不能誘導(dǎo)產(chǎn)生相應(yīng)的中和抗體,故SP70和SP55不能用于EV71多肽疫苗的研發(fā)。
[Abstract]:In recent years, hand foot and mouth disease (HFMD) has been a persistent epidemic in China, and death cases are increasing year by year. The HFMD patients caused by enterovirus 71 (EV71) infection may lead to severe complications, such as aseptic meningitis, brainstem encephalitis and poliomyelitis like paralysis, and may also cause more severe pulmonary water. Severe cases of swelling, accompanied by pulmonary edema, are developing rapidly and with high mortality. At present, the molecular mechanism of EV71 is not clear, and there is no specific drug and vaccine against this virus. This study explores the relevant rules of the EV71 antibody response from the humoral immune angle, in order to provide the pathogenesis of the EV71 epidemic, treatment and vaccine research and development. Some theoretical references. There are two aspects of this study. First, the antibody cross reactivity between EV71 and poliovirus (PV) is explored by clinical serum samples and experimental animal levels, and second, the study of SP70 and SP55 in EV71 infected people through the enzyme linked immunosorbent assay ELISA experiment and neutralization inhibition test Whether or not the body can induce the corresponding neutralization antibody.
ELISA detected the responsiveness of EV71 or PV to the serum IgG of healthy children. The study found that EV71 and PV positive EV71 negative healthy children had a high responsiveness (positive rate of 77.8%), and there was a positive correlation (r=0.58) in the IgG of these two viruses in these PV positive EV71 negative children's serum (r=0.58). Some PV positive children could not neutralize EV71.EV71 and PV positive, but EV71 negative sera were incubated to infect human mononuclear leukemia cell line (THP-1). After infection, 24 hours after infection, the number of EV71 virus entered THP-1 by real-time quantitative PCR detection. The results showed that the serum of PV positive EV71 negative children could enhance EV71 infection into THP-1 cells. It is preliminarily indicated that PV vaccination can induce antibodies that produce cross reactivity with EV71, although these cross reactive antibodies are non neutralizing antibodies, but these non neutralizing antibodies can promote EV71 infection in THP-1 in vitro, which suggests that PV inoculated with EV71 cross reacting antibodies may be associated with the pathogenesis of EV71, so this study may be EV71 The mechanism of pathogenesis has opened up a new direction.
In order to further verify the cross reactivity of antibody between EV71 and PV at the level of experimental animals with clear background, the capsid protein VP1 and non structural protein 3C of EV71 and PV were obtained by prokaryotic expression and affinity purification, respectively. The immunization of PV-VP1, EV71-VP1, EV71 inactivated vaccine and PV inactivation vaccine (IPV) was obtained by subcutaneous immunization of BALB/c mice, respectively. Serum immunoblotting showed that PV-VP1 could react with EV71-VP1 antiserum, and on the contrary, EV71-VP1 could react with PV-VP1 antiserum; competitive ELISA test further showed that EV71-VP1 protein could obviously inhibit the combination of PV-VP1 and its specific immune sera antibody, and vice versa; but microneutralization test revealed that the serum of PV positive mice could not be found. Neutralizing EV71.SPF mice showed that there was a large number of cross reactive non neutralizing antibodies between EV71 and PV.
The important linear neutralization epitopes (SP70 and SP55) of two EV71 were detected by previous mice. In order to study the reactivity of these two epitopes in the human body, the two epitopes were synthesized, the serum of EV71 infected children and the serum of healthy children who had not been infected with EV71 were collected, and the serum IgG of SP70 or SP55 and EV71 infected patients was detected by ELISA. There was no difference in the reactivity of serum IgG between SP70 or SP55 and EV71 infected persons and the reactivity of serum IgG in healthy children without infection. The serum.EV71 positive children were neutralized in vitro and EV71 after pre incubation with SP70 or SP55. The study found that these two peptides did not inhibit the neutralization effect of EV71 positive human serum. Obviously, SP70 and SP55 can not induce corresponding neutralizing antibodies in human body when natural infection occurs, so SP70 and SP55 can not be used for the development of EV71 peptide vaccine.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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