手足口病预防性基因工程重组疫苗的研究

发布时间：2018-06-29 17:42

本文选题：HbcAg + EV71　；参考：《北京工业大学》2014年博士论文

【摘要】：肠道病毒71型（enterovirus71, EV71）与柯萨奇A16病毒是引起手足口病（hand foot and mouth disease, HFMD）的主要病原体。近年来手足口病在亚太地区的多次爆发，使当地青少年儿童的身体健康受到严重威胁。虽然手足口病发病后病情较温和且具有一定的自愈性，但如任由其发展也将导致严重的神经系统并发症，更为严重者可导致死亡。我国卫生部于2008年将手足口病列为法定报告的丙类传染病。迄今为止，防治手足口病的相关疫苗仍未上市。因此，研发广谱高效的手足口病疫苗意义重大。 EV71病毒是一种非包膜的RNA病毒，隶属于小RNA病毒科（Picornaradae）家族，其颗粒的直径大小约30nm左右。EV71病毒的颗粒呈二十面体立体对称结构；其病毒衣壳由60组多聚蛋白所组成，每组多聚蛋白均含有4种衣壳蛋白（VP1-VP4）。有证据显示VP1-VP3蛋白位于正二十面体核衣壳的外表面，而VP4蛋白位于核衣壳的内部与RNA核心紧密连接。VP4蛋白长约70个氨基酸，N端豆蔻酰化。晶体结构X光衍射图分析表明：成熟的EV71病毒颗粒其立体结构与其他肠道病毒极其相似。本文的研究首先将EV71病毒C4亚型VP4蛋白N端前20个氨基酸的基因融合入乙肝病毒核心抗原（HbcAg）基因中，融合后的重组基因克隆至pET-22b (+)载体中。利用大肠杆菌表达系统分别表达HBc-N149蛋白以及HBc-N149-VP4-N20蛋白。IPTG诱导目的蛋白表达后利用Western-blot检测蛋白表达情况。诱导表达后的目的蛋白利用镍柱进行纯化。电镜观察结果显示，HBc-N149蛋白与HBc-N149-VP4-N20蛋白均可有效形成病毒样颗粒，且直径约为25-30nm。这一结果表明，将EV71病毒VP4蛋白N端前20个氨基酸插入HbcAg蛋白后并未改变其自组装形成病毒样颗粒的特性。为了研究重组HbcAg蛋白是否能产生针对VP4-N20的抗体，将纯化后的HBc-N149蛋白以及HBc-N149-VP4-N20蛋白通过肌肉注射的方式免疫雌性BALB/c小鼠。阴性对照组免疫PBS溶液。实验动物免疫后收集血清进行血清学相关检测。实验结果显示，重组HbcAg蛋白可以产生针对VP4-N20的抗体。为了验证包含有VP4-N20的重组HbcAg蛋白是否能产生针对EV71病毒的中和抗体，利用实验动物免疫后收集的血清进行体外中和实验。实验结果显示，HBc-N149-VP4-N20蛋白免疫组血清可以中和EV71病毒；HBc-N149蛋白免疫组血清未能中和EV71病毒。这一结果表明含有VP4-N20的重组HbcAg蛋白免疫实验动物后产生的抗体具有针对EV71病毒的中和活性。为了进一步验证抗HBc-N149-VP4-N20蛋白的血清是否能为实验小鼠提供针对EV71病毒的保护能力，进行了小鼠体内攻毒实验。实验动物选择1日龄BALB/c乳鼠。实验中选用针对乳鼠具有较高毒力的EV71病毒BrCr-TR株，通过腹腔注射病毒-血清混合物（血清分别选用免疫HBc-N149-VP4-N20蛋白的血清以及免疫HBc-N149蛋白的血清）、病毒-PBS溶液混合物。实验进行至第7天时，病毒-HBc-N149蛋白血清组乳鼠、病毒-PBS溶液组乳鼠开始出现行动迟缓、四肢乏力、四肢瘫痪等病症，有甚者出现死亡症状；第16天即实验结束时，病毒-HBc-N149蛋白血清组乳鼠存活率为40%，病毒-PBS溶液组乳鼠存活率为20%。反之，，病毒-HBc-N149-VP4-N20蛋白血清组乳鼠存活率为90%。这一结果表明，重组HbcAg颗粒即HBc-N149-VP4-N20蛋白的免疫血清可以针对EV71病毒为新生乳鼠提供保护效力。进一步的研究中，利用肽库筛查实验确定了VP4-N20蛋白产生中和抗体的抗原决定簇表位。实验中肽库的设计原则为，分别从VP4-N20蛋白N端以及C端每次减少两个氨基酸。实验结果显示，当VP4-N20蛋白N端减少至第6个氨基酸时，或者C端减少至第10个氨基酸时，针对VP4-N20蛋白的免疫血清将不再和VP4-N20蛋白相结合。这一实验结果表明，VP4-N20蛋白产生中和抗体的抗原决定簇表位为其N端减少6个氨基酸、C端减少10个氨基酸后所剩多肽序列。本文的研究中，将EV71病毒C4亚型的VP4蛋白N端前20个氨基酸融合入HbcAg中且在大肠杆菌中表达。实验结果显示，融合目的蛋白在表达后可以自组装形成嵌合式病毒样颗粒，并且可以诱导产生针对病毒的有效中和抗体。后续试验中，通过肽库筛查确定了这一序列的抗原决定簇位点所在。实验结果最终表明，EV71病毒VP4蛋白中和表位的鉴定为研制广谱抗EV71预防性疫苗提供了新的思路和线索。
[Abstract]:Enterovirus71 (enterovirus71, EV71) and Coxsackie A16 virus are the main pathogens causing hand foot and mouth disease (hand foot and mouth disease, HFMD). In recent years, hand foot and mouth disease (HFMD) has erupted many times in the Asia Pacific region, causing serious threat to the health of young children in the region. Self healing, but the development of it will lead to serious neurological complications, and more serious people can lead to death. In 2008, the Ministry of health of China listed hand foot and mouth disease as a legal report of the class C infectious disease. So far, the related vaccine against hand foot and mouth disease has not been listed. Therefore, the significance of developing a broad spectrum and efficient hand foot and mouth disease vaccine is developed. It's important.
EV71 virus is a non enveloped RNA virus, belonging to the family of small RNA virus family (Picornaradae). The particle size of about 30nm.EV71 virus particles is twenty dimensional symmetry structure; its viral capsid is composed of 60 groups of polyproteins and each group of polyproteins contains 4 kinds of capsid protein (VP1-VP4). Evidence shows VP1-VP3 The protein is located on the outer surface of the nucleocapsid of the positive twenty body, while the VP4 protein is located inside the nucleocapsid and the RNA core closely connected with the.VP4 protein, about 70 amino acids long and N terminal myrisylation. The crystal structure X light diffraction analysis shows that the stereoscopic structure of the mature EV71 virus particles is extremely similar to the other enteroviruses.
This study first fused the 20 amino acids of the EV71 virus C4 subtype VP4 protein into the hepatitis B virus core antigen (HbcAg) gene, and cloned the recombinant gene into the pET-22b (+) vector. The expression system of the Escherichia coli expression system was used to express the HBc-N149 protein and the HBc-N149-VP4-N20 protein.IPTG to induce the expression of the target protein. The protein expression was detected by Western-blot. The purified target protein was purified by the nickel column. The results showed that the HBc-N149 protein and HBc-N149-VP4-N20 protein could effectively form the virus like particles, and the diameter of the protein was about 25-30nm.. The results showed that the 20 amino acids of the N end of the EV71 virus VP4 protein were inserted into HbcAg eggs. The characteristics of self assembling virus like particles did not change after white.
In order to study whether the recombinant HbcAg protein could produce antibodies against VP4-N20, the purified HBc-N149 protein and HBc-N149-VP4-N20 protein were immunized by intramuscular injection of the female BALB/c mice. The negative control group was immunized with PBS solution. The serum was collected from the experimental animal after immunization and the serological correlation was detected. The experimental results showed that the recombinant HbcAg was reconstituted. The protein can produce antibodies against VP4-N20.
In order to verify whether the recombinant HbcAg protein containing VP4-N20 could produce neutralizing antibodies against EV71 virus, the serum collected from the experimental animal immune system was used to neutralize the neutralization test in vitro. The results showed that the serum of HBc-N149-VP4-N20 protein immune group could neutralize the EV71 virus, and the serum of HBc-N149 protein immune group did not neutralize the EV71 virus. The results showed that the recombinant HbcAg protein containing VP4-N20 immunized with experimental animals had neutralizing activity against EV71 virus.
In order to further verify whether the serum of anti HBc-N149-VP4-N20 protein could provide the protective ability of EV71 virus for experimental mice, the experiment was carried out in mice. The experimental animals selected 1 day old BALB/c milk mice. In the experiment, the EV71 virus BrCr-TR strain with high virulence was selected and the virus serum mixture was injected into the abdominal cavity. The serum of the immune HBc-N149-VP4-N20 protein and the serum of the immune HBc-N149 protein, and the mixture of the -PBS solution of the virus, were used in the serum, respectively. When the experiment was carried out for seventh days, the virus -HBc-N149 protein serum group of milk mice, the virus -PBS solution group of milk mice began to appear to be slow in action, fatigue in the extremities, limbs paralysis and so on. At the end of the 16 day, the survival rate of the virus -HBc-N149 protein serum group was 40%, the survival rate of the virus -PBS solution group was 20%. on the contrary, the survival rate of the virus -HBc-N149-VP4-N20 protein serum group was 90%.. The result showed that the immune serum of the recombinant HbcAg granule, HBc-N149-VP4-N20 protein, could be a new milk rat against EV71 virus. Provide protection effectiveness.
In the further study, the peptide epitope of the neutralizing antibody produced by VP4-N20 protein was determined by the peptide library screening test. The design principle of the peptide library in the experiment was to reduce two amino acids each time from the VP4-N20 protein N end and the C terminal respectively. The experimental results showed that when the N end of the VP4-N20 protein was reduced to sixth amino acids, or the C end was reduced to the first. At 10 amino acids, the immune sera against VP4-N20 protein will no longer be combined with VP4-N20 protein. This experimental result shows that the epitope of the antigenic determinant of the neutralizing antibody produced by VP4-N20 protein reduces 6 amino acids for its N terminal, and the C terminal reduces the sequence of polypeptide after 10 amino acids.
In this study, the 20 amino acids of the VP4 protein N terminal of the VP4 protein of the C4 subtype of the virus were fused into HbcAg and expressed in Escherichia coli. The results showed that the fusion target protein could be self assembled to form chimeric virus like particles after the expression, and could induce effective neutralizing antibodies against the virus. In the follow-up test, the peptide library was used. The test results showed that the identification of the neutralization epitopes of the EV71 virus VP4 protein provided new ideas and clues for the development of the broad-spectrum anti EV71 preventive vaccine.
【学位授予单位】：北京工业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.5

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