重配H1N1亚型流感病毒减毒疫苗株的研究

发布时间：2018-05-30 03:43

本文选题：流感病毒 + H1N1亚型　；参考：《中国人民解放军军事医学科学院》2007年博士论文

【摘要】： 流感是由流感病毒(influenza virus)引起的一种以侵害呼吸系统为主的疾病，在世界各国广泛流行，是危害人类健康的重要传染病之一，因此，WHO宣布对流感要严加监控。20世纪曾有过3次全球性的流感大流行，仅第一次大流行(1918～1919年)就造成2000多万人死亡，比第一次世界大战死亡的总人数还要多，而且死亡的大多数是青壮年，造成巨大的社会和经济损失。据国家权威部门透露，我国每年流感的发病高达10亿人次以上，经济损失数百亿元以上。近些年来，我国的生态环境严重破坏，人流感的发生和发展规律发生了较大变化，发病频率上升，造成了严重的社会影响和重大的经济损失。流感病毒属于正粘病毒科(Orthomyxoviridae)，是一种含有8个不同基因片段的分节段RNA病毒，分为甲(A)、乙(B)、丙(C)三型，其中A型流感病毒对人类危害最严重，它引起的并发症危害大，造成神经-内分泌-免疫网络的紊乱，已严重影响人类健康和国家经济建设。A型人流感病毒自发现以来，曾出现过三种亚型：1918～1919年西班牙流感流行的是H1N1亚型；1957年之前H2N2亚型出现，而H1N1亚型消失了；1968年H3N2亚型出现，而H2N2至今没有出现；1977年H1N1再次出现，而至今H1N1亚型仍是流行主型，它引起的流感大流行对人类社会的严重危害性和公共卫生意义都受到关注。编码流感病毒的抗原主要有血凝素蛋白(Hemagglutinin，HA)、神经氨酸酶(Neuraminidase，NA)、核蛋白(Nuclear Protein，NP)和基质蛋白M(Matrix2)等多肽分子。由于HA、NA高度变异决定着病原体的特殊性，致使每年都有新的病毒株出现，给流感的防控带来了难题。目前，对于流感尚无有效的治疗手段，而疫苗免疫接种发挥着重要的作用。流感疫苗经过五十多年的发展，主要有灭活全病毒疫苗、裂解疫苗、亚单位疫苗、DNA疫苗和减毒活疫苗。由于流感病毒的抗原变异性大，使得疫苗生产每年都需要更换病毒株。当前使用的注射三价灭活疫苗有效，但在保护效果，特别是交叉免疫保护和接种策略上还不理想；亚单位疫苗使用安全，主要针对HA、NA抗原，有一定的免疫保护效果，但是制备周期长，对于季节性流感来说不太实用，并且保护谱不全面；DNA疫苗起步较晚，主要针对某一保护性抗原成分设计，仍然是保护不全面，另外抗原用量大、特别是潜在基因整合等瓶颈问题：而传统的减毒活疫苗可以诱导包括细胞免疫、体液免疫和局部免疫在内的较全面的免疫效应，但仍保留一定残余毒力，存在潜在的“返祖”现象，因此迫切需要发展新的疫苗研制手段。近年来，反向遗传技术的诞生为减毒活疫苗的发展提供了良好的契机。目前，基于RG(reverse genetics，RG)技术的流感减毒活疫苗成为流感疫苗发展的方向，1999年Neumann等、Hoffmann等学者建立了完全以质粒为基础的流感病毒反向遗传技术，为流感减毒活疫苗的研制提供了新的思路。以冷适应减毒病毒株为背景，拯救重配的流感减毒活疫苗株，进而研发新一代的流感减毒活疫苗是当今流感疫苗研究的热点。本研究采用反向遗传技术，通过8质粒流感病毒拯救系统，以冷适应流感病毒株A／Ann Arbor／6／60(H2N2)的6个内部基因为病毒骨架，与2006～2007年流感病毒株A／New Caledonia／20／99(H1N1)的HA和NA基因重排，建立了重配冷适应H1N1亚型流感病毒株拯救体系，为流感减毒活疫苗的研发开辟了新的思路，同时为流感的安全、有效免疫预防提供研制策略和理论依据。主要研究内容包括：一、双向转录／表达载体pAD3000的构建要想得到拯救成功的重配流感病毒株，必须要有一套完善的拯救体系，而在拯救体系中，表达载体起着至关重要的作用。为了使得转染质粒在拯救系统中能够高表达，提高其在细胞中的转录翻译水平，我们对载体pHW2000进行了改造，根据转染所用细胞的类型，选择用SV40的多聚A(polyA)信号序列取代牛生长激素(BGH)的多聚腺苷酸信号序列，以期获得高效的转染效率和病毒拯救效率，为流感减毒活疫苗的制备提供理论基础。根据相关文献报道，利用pcDNA3.1(+)质粒为模板，扩增得到了SV40的多聚A(polyA)信号序列，该片段大小为138bp。同时从国外得到的双向转录／表达载体pHW2000，是在pcDNA3.0的基础上经过一系列的改造而演化而来的，在该载体的基础上，我们将其升级为双向转录／表达载体pAD3000，通过PCR扩增和测序验证基因序列是完全正确的，并且利用序列分析软件对其全序列进行了分析。以上工作为流感病毒8质粒系统转录／表达载体的构建奠定了基础。二、流感病毒8质粒系统的构建利用构建好的双向转录／表达载体pAD3000载体系统，我们选择冷适应、减毒的流感病毒株A／Ann Arbor／6／60的6个内部基因(PB2、PB1、PA、NP、M、NS)作为拯救病毒骨架，人工合成其基因全序列，同时引入PB1-391E，581G，661T，PB2-265S，NP-34G5个氨基酸的突变位点，根据已发表的序列设计扩增6个基因的特异引物对。将2006～2007年WHO宣布的H1N1亚型流感病毒疫苗株A／New Caledonia／20／99，接种10～11日龄SPF鸡胚增殖，通过蔗糖密度梯度离心纯化病毒，提取基因组总RNA，反转录成cDNA。根据Hoffmann等发表的通用引物序列扩增HA和NA基因并测序。为获得准确的5’和3’端序列，在PCR过程中采用高保真聚合酶，且挑选8～10个克隆测序，保证序列的准确性。经序列分析软件比对，得到拼接出含5’和3’端非编码区的8个基因的全长序列。设计带有BsmBⅠ、BsaⅠ或AarⅠ酶切位点的引物扩增A／Ann Arbor／6／60的6个内部基因片段和A／New Caledonia／20／99的HA和NA基因，分别克隆入pAD3000，并进行了测序验证，从而构建了8个polⅠ-polⅡ系统的转录／表达质粒，分别命名为pMDV-A-PB2，pMDV-A-PB1，pMDV-A-PA，pMDV-A-NP,pMDV-A-M，pMDV-A-NS，pMDV-A-HA，pMDV-A-NA，以期通过同一个载体、转染后利用细胞中的polⅠ和polⅡRNA聚合酶、实现vRNA和mRNA的转录和表达，为冷适应H1N1亚型流感病毒减毒株的构建提供了保证。三、重配H1N1亚型流感病毒减毒株的制备 1、冷适应流感病毒拯救系统的验证用A／New Caledonia／20／99的2个表面基因(HA和NA)或冷适应病毒株A／Ann Arbor／6／60的任意一个内部基因，而其它内部基因来自A／PR／8／34，进行所有组合的基因重排，即8种7+1和1种6+2组合形式，将相应的转录／表达质粒组合，共转染COS-1细胞，均产生了预期组合、有感染性的H1N1亚型流感病毒，表明构建的8个转录／表达载体均能有效工作，且冷适应病毒株的6个内部病毒骨架能够协同发挥作用，为进一步筛选和构建重配冷适应流感病毒株奠定了基础。同时用整合A／PR／8／34的8个基因的重组质粒作阳性对照，也产生了转染子病毒。在这个过程中，优化共转染体系，在35mmdish上即可产生足以直接用鸡胚增殖的子代病毒。对7+1组合的PB2／PR8、PR8转染子代病毒及6+2组合的PR8／rMDV-A进行了初步鉴定和部分生物学特性分析。经电镜观察，，转染子病毒的形态与野生型流感病毒相似。RT-PCR表明引入的氨基酸突变在子代病毒中存在。HA、HI和IFA(indirect immunofluorecent assay，IFA)显示有重配病毒产生。PR8／rMDV-A的PFU结果表明低温如33℃更利于重配病毒的繁殖，经过鸡胚传代和MDCK感染实验，PR8／rMDV-A病毒能在鸡胚中高滴度增殖，感染鸡胚的能力强，这与提供表面基因的A／PR／8／34病毒株相似，而且对鸡胚的毒力弱、低温利于病毒增殖这又与提供6个内部基因的A／Ann Arbor／6／60相似。以上这些实验结果证实了冷适应病毒拯救系统的有效性。 2、H1N1流感病毒减毒疫苗株的拯救及鉴定利用验证好的冷适应的流感病毒6个质粒系统，作为拯救流感病毒的骨架，与构建好的2006～2007年流感病毒株A／New Caledonia／20／99(H1N1)的HA和NA基因进行重排，8个重组质粒共转染COS-1细胞，成功得到了重配H1N1亚型流感减毒病毒株，拯救的流感减毒株在第一代鸡胚尿囊液中病毒效价为1:512～1:1024，连续传4代之后，发现病毒效价稳定，各代之间HA和NA基因的同源性达99.9％以上，经电镜观察发现重配病毒的形态与野生流感病毒株没有明显差别，并对其生物学特性进行了初步的鉴定，深入的研究工作还在进行之中。以上建立的反向遗传系统重配冷适应H1N1亚型流感病毒株，为流感病毒减毒活疫苗的研制和黏膜免疫机制研究奠定了坚实的基础。
[Abstract]:Influenza is a disease caused by influenza virus (influenza virus), which is the main disease that violates the respiratory system. It is widely popular in the world and is one of the important infectious diseases that harm human health. Therefore, WHO announced that the influenza should be strictly monitored in the.20 century and there have been 3 global influenza pandemics in the century, only the first pandemic (1918~1919 years). The death of about 20000000 people is more than the total number of deaths in World War I, and most of the deaths are young and young, causing huge social and economic losses. According to the state authority, the incidence of influenza in China is more than 1 billion times a year, and the economic loss is hundreds of billions of yuan. In recent years, the ecological environment of our country is strict. Heavy damage, the occurrence and development of human flu has undergone great changes, the frequency of the disease has increased, resulting in serious social impact and major economic losses.
Influenza virus belongs to the family of Orthomyxoviridae. It is a segmental RNA virus containing 8 different gene fragments. It is divided into a (A), B (B), and C (C) three. Among them, the A influenza virus is most harmful to human beings. It causes great complications and the disorder of the neuroendocrine immune network, which has seriously affected human health and human health. Since the discovery of the.A type human influenza virus in the national economy, there have been three subtypes since the outbreak of the H1N1 influenza in 1918~1919 years; the H2N2 subtype appeared before 1957, and the H1N1 subtype disappeared; the H3N2 subtype appeared in 1968, and the H2N2 did not appear so far; H1N1 appeared again in 1977, and so far H1N1 subtype is still popular. The influenza pandemic caused by it is concerned about the serious harm and public health significance of human society. The antigen encoding influenza viruses mainly include Hemagglutinin (HA), Neuraminidase (NA), Nuclear Protein, NP, and matrix protein M (Matrix2). Because HA, NA is changed. The difference determines the specificity of the pathogen, resulting in the emergence of new strains of virus every year, which brings difficulties to the prevention and control of influenza.
At present, there is no effective treatment for influenza, and vaccine immunization plays an important role. After more than 50 years of development, influenza vaccine includes inactivated whole virus vaccine, lysis vaccine, subunit vaccine, DNA vaccine and live attenuated vaccine. The current use of the injection trivalent inactivated vaccine is effective, but it is not ideal for the protection effect, especially in the cross immunization protection and vaccination strategy; subunit vaccine is safe to use, mainly for HA, NA antigen, and has a certain immune protection effect, but the preparation cycle is long, for seasonal influenza is not very practical, and protection spectrum. Not comprehensive; the DNA vaccine started late, mainly aimed at the design of a protective antigen, still the bottleneck problem of incomplete protection and large amount of antigen, especially the potential gene integration, and the traditional attenuated live vaccine can induce more comprehensive immune effects including cell immunity, humoral immunity and local immunization, but still still have a more comprehensive immune effect. In recent years, the birth of reverse genetic technology has provided a good opportunity for the development of live attenuated vaccine. At present, the live attenuated influenza vaccine based on RG (reverse genetics, RG) technology has become the direction of influenza vaccine development, 19 99 years, such as Neumann, and so on, Hoffmann and other scholars have established a completely plasmid based reverse genetic technology for influenza virus, which provides a new idea for the development of a live attenuated influenza vaccine. To save the redistributed live attenuated vaccine strain with the background of cold adaptation attenuated virus strain, and then develop a new generation of live attenuated influenza vaccine as a current influenza vaccine. In this study, the reverse genetic technique was used to establish a heavy colder adaptation to H1N1 Asia by using the 8 plasmid influenza virus rescue system and 6 internal genes of influenza virus strain A / Ann Arbor / 6 / 60 (H2N2) as the virus skeleton, and the HA and NA gene rearrangements of the 2006~2007 year influenza virus strain A / New Caledonia / 20 / 99 (H1N1). The rescue system of influenza virus has opened up a new idea for the development of the live attenuated influenza vaccine, and provides the development strategy and theoretical basis for the safety of influenza and effective immunization prevention. The main contents include:
The construction of bi-directional transcriptional / expression vector pAD3000
In order to save the successful strain of the influenza virus, we must have a perfect rescue system, and in the rescue system, the expression vector plays a vital role. In order to make the transfection plasmid high expression in the rescue system and improve its transcriptional translation level in the cell, we reformed the carrier pHW2000, based on the transformation. In order to obtain efficient transfection efficiency and rescue efficiency of virus, the SV40 (polyA) signal sequence was selected to replace the polyadenylate signal sequence of bovine growth hormone (BGH) in order to provide a theoretical basis for the preparation of the live attenuated influenza vaccine. According to phase Guan Wenxian, the pcDNA3.1 (+) plasmid was used as a template. The sequence of SV40's polycondensation A (polyA) signal, which is the size of 138bp. and the bi-directional transcriptional / expression vector pHW2000 from abroad, is evolved on the basis of a series of modifications on the basis of pcDNA3.0. On the basis of the vector, we upgrade it to a dual transcriptional / expression vector pAD3000, which is amplified and sequenced by PCR. The gene sequence was completely correct and the sequence analysis software was used to analyze the whole sequence. The above work laid the foundation for the construction of the transcription / expression vector of the influenza virus 8 plasmid system.
Two, construction of the 8 plasmid system of influenza virus
Using the constructed bi-directional transcriptional / expression vector pAD3000 vector system, we select 6 internal genes (PB2, PB1, PA, NP, M, NS) of influenza virus strain A / Ann Arbor / 6 / 60, which are cold adapted and attenuated influenza virus strain (PB2, PB1, PA, NP, M, NS). The specific primers of 6 genes were designed according to the published sequence. The H1N1 subtype influenza virus vaccine strain A / New Caledonia / 20 / 99, declared in 2006~2007 year WHO, was inoculated with 10~11 day old SPF chicken embryos, the virus was purified by sucrose density gradient centrifugation, and the total RNA of the gene group was extracted, and the reverse transcriptional cDNA. was published according to Hoffmann and so on. The HA and NA genes were amplified and sequenced by the universal primer sequence. In order to obtain accurate 5 'and 3' end sequences, high fidelity polymerase was used in the PCR process and 8~10 clones were selected to ensure the accuracy of the sequence. Through sequence analysis software comparison, a full length sequence of 8 genes with 5 'and 3' uncoded regions was obtained. Design with B SmB I, Bsa I or Aar I primers amplified 6 internal gene fragments of A / Ann Arbor / 6 / 60 and HA and NA genes of A / New Caledonia / 20 / 99, respectively, and were cloned into pAD3000. A, pMDV-A-NP, pMDV-A-M, pMDV-A-NS, pMDV-A-HA, pMDV-A-NA, in order to realize the transcription and expression of vRNA and mRNA by using the same carrier, transfection, Pol I and Pol II RNA polymerase in the cells, to provide a guarantee for the construction of the cold adaption of the virus strain of the influenza virus.
Three, preparation of a strain of H1N1 subtype influenza virus
1, verification of cold adaptation to influenza virus rescue system
With A / New Caledonia / 20 / 99 of 2 surface genes (HA and NA) or cold adapted virus strain A / Ann Arbor / 6 / 60 of any internal gene, and other internal genes from A / PR / 8 / 34, all combinations of gene rearrangement, that is, 8 7+1 and 1 types of 6+2 combinations, combined the corresponding transcriptional / expression plasmids and co transfected the cells. The expected combination, an infectious H1N1 subtype influenza virus, indicates that the 8 transcriptional / expression vectors can work effectively, and the 6 internal virus skeletons of the cold adapted virus strain can play a synergistic role. It lays the foundation for further screening and construction of the cold adapted influenza virus strains. Simultaneously, the integration of A / PR / 8 / 34 is used. The recombinant plasmid of the 8 genes was used as a positive control, and the transfection virus was produced. In this process, the co transfection system was optimized to produce a progeny virus that was sufficient to proliferate directly with the chicken embryo on 35mmdish.
PB2 / PR8, PR8 transfected progeny virus and 6+2 combination PR8 / rMDV-A were preliminarily identified and some biological characteristics were analyzed. By electron microscopy, the morphology of the transfected subvirus was similar to that of the wild type influenza virus (.RT-PCR), which showed that the amino acid mutation introduced in the virus existed.HA, HI and IFA (indirect) in the offspring virus. Y, IFA) showed that the PFU results of.PR8 / rMDV-A produced by the recombinant virus showed that low temperature, if 33, was more beneficial to the reproduction of the redistribution virus. Through the chicken embryo transfer and MDCK infection experiment, the PR8 / rMDV-A virus could proliferate in the chicken embryo, and the ability to infect the chicken embryo was strong, which was similar to the A / PR / 8 / 34 virus strain which provided the surface base, and to the chicken embryo. The virulence was weak, and the low temperature was beneficial to the proliferation of the virus, which was similar to the A / Ann Arbor / 6 / 60, which provided 6 internal genes. These experimental results confirmed the effectiveness of the cold adapted virus rescue system.
2, rescue and identification of H1N1 influenza virus attenuated vaccine strain
6 plasmid systems of cold adapted influenza virus were used as the skeleton of the influenza virus to save the influenza virus. It was rearranged with the HA and NA genes of the constructed 2006~2007 year influenza virus strain A / New Caledonia / 20 / 99 (H1N1), and 8 recombinant plasmids were co transfected to COS-1 cells. The virus titer of the strain of influenza virus was 1:512 ~ 1:1024 in the first generation chicken embryo allantoic fluid. After 4 successive generations, the virus titer was stable and the homology of HA and NA genes between each generation was more than 99.9%. The morphology of the redistribution virus was found to have no obvious difference with the wild influenza virus strain by electron microscopy, and the biological characteristics were preliminarily studied. The further research work is still in progress. The above established reverse genetic system rematches cold adapted to the H1N1 subtype influenza virus strain, which lays a solid foundation for the development of the live attenuated influenza virus vaccine and the study of the mucosal immune mechanism.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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