肠道病毒71型抗体反应规律初步研究

发布时间：2018-05-05 13:25

本文选题：肠道病毒71型 + 脊髓灰质炎病毒　；参考：《华东师范大学》2012年硕士论文

【摘要】：近年来,手足口病(HFMD)在我国呈持续性流行态势,而且死亡病例逐年增加。肠道病毒71型(EV71)感染所引起的HFMD患者易导致严重的并发症,如无菌性脑膜炎、脑干脑炎和脊髓灰质炎样的麻痹等神经系统相关疾病,还可能引起更严重的肺水肿,伴有肺水肿的重症病例病情进展快,死亡率高。目前,EV71致病的分子机制尚不清楚,而且没有针对这种病毒的特异性药物及疫苗。本研究从体液免疫角度探索EV71抗体反应的有关规律,以期为EV71流行病的致病机理、治疗及疫苗研发提供一些理论参考。本项研究有两方面的内容：其一,通过临床血清样本研究及实验动物水平的验证,探讨EV71与脊髓灰质炎病毒(PV)之间的抗体交叉反应性；其二,通过酶联免疫吸附试验ELISA实验及中和抑制实验研究SP70和SP55在EV71感染的人体内能否诱导产生相应的中和抗体。 ELISA检测EV71或PV与健康儿童血清IgG的反应性,研究发现EV71与PV阳性EV71阴性健康儿童血清IgG有很高的反应性(阳性率达77.8%),而且在这些PV阳性EV71阴性儿童血清中针对这两种病毒的IgG在含量上呈正相关(r=0.58)。微量中和试验研究发现,这些PV阳性儿童血清不能中和EV71。EV71与PV阳性但EV71阴性人血清孵育后感染人单核白血病细胞系(THP-1),感染后培养24小时,以实时定量PCR检测进入THP-1的EV71病毒数量,结果发现PV阳性EV71阴性的儿童血清能增强EV71感染进入THP-1细胞。以上结果初步表明,PV疫苗接种能诱导产生与EV71交叉反应性的抗体,虽然这些交叉反应性抗体是非中和抗体,但这些非中和抗体在体外能促进EV71感染THP-1,这表明PV接种产生的与EV71交叉反应性抗体可能与EV71的致病有关,所以本研究可能为EV71致病机制的研究开辟一个可能的新方向。为了在背景清晰的实验动物水平进一步验证EV71与PV之间的抗体交叉反应性,以原核表达与亲和纯化等方法分别获得EV71和PV的衣壳蛋白VP1及非结构蛋白3C,将PV-VP1、EV71-VP1、EV71灭活疫苗及PV灭活疫苗(IPV)分别经皮下免疫BALB/c小鼠制得免疫血清。蛋白免疫印迹实验显示PV-VP1能与EV71-VP1抗血清反应,反之,EV71-VP1也能与PV-VP1抗血清反应；竞争ELISA试验进一步显示EV71-VP1蛋白能明显抑制PV-VP1与其特异性免疫血清抗体的结合,反之亦然；但微量中和实验揭示PV阳性小鼠血清不能中和EV71。SPF级小鼠实验研究证实EV71与PV之间确实存在大量交叉反应性非中和抗体。前人以小鼠实验研究发现两个EV71的重要线性中和表位(SP70与SP55)。为了研究这两个表位在人体内的反应性,合成这两个表位,收集EV71感染儿童恢复期血清及未曾感染EV71的健康儿童血清,以ELISA检测SP70或SP55与EV71感染患者恢复期血清IgG的反应性。研究发现,SP70或SP55与EV71感染者血清IgG的反应性和与未曾感染的健康儿童血清IgG的反应性没有差异。EV71阳性儿童血清分别与SP70或SP55预孵育后在体外中和EV71,研究发现这两个肽不能抑制EV71阳性人血清的中和效应。本研究表明,在自然感染时,SP70和SP55在人体内不能诱导产生相应的中和抗体,故SP70和SP55不能用于EV71多肽疫苗的研发。
[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.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【相似文献】