本文選題：流感病毒 + H1N1亞型　； 參考：《中國人民解放軍軍事醫(yī)學科學院》2007年博士論文

【摘要】： 流感是由流感病毒(influenza virus)引起的一種以侵害呼吸系統(tǒng)為主的疾病，在世界各國廣泛流行，是危害人類健康的重要傳染病之一，因此，WHO宣布對流感要嚴加監(jiān)控。20世紀曾有過3次全球性的流感大流行，僅第一次大流行(1918～1919年)就造成2000多萬人死亡，比第一次世界大戰(zhàn)死亡的總人數還要多，而且死亡的大多數是青壯年，造成巨大的社會和經濟損失。據國家權威部門透露，我國每年流感的發(fā)病高達10億人次以上，經濟損失數百億元以上。近些年來，我國的生態(tài)環(huán)境嚴重破壞，人流感的發(fā)生和發(fā)展規(guī)律發(fā)生了較大變化，發(fā)病頻率上升，造成了嚴重的社會影響和重大的經濟損失。 流感病毒屬于正粘病毒科(Orthomyxoviridae)，是一種含有8個不同基因片段的分節(jié)段RNA病毒，分為甲(A)、乙(B)、丙(C)三型，其中A型流感病毒對人類危害最嚴重，它引起的并發(fā)癥危害大，造成神經-內分泌-免疫網絡的紊亂，已嚴重影響人類健康和國家經濟建設。A型人流感病毒自發(fā)現(xiàn)以來，曾出現(xiàn)過三種亞型：1918～1919年西班牙流感流行的是H1N1亞型；1957年之前H2N2亞型出現(xiàn)，而H1N1亞型消失了；1968年H3N2亞型出現(xiàn)，而H2N2至今沒有出現(xiàn)；1977年H1N1再次出現(xiàn)，而至今H1N1亞型仍是流行主型，它引起的流感大流行對人類社會的嚴重危害性和公共衛(wèi)生意義都受到關注。編碼流感病毒的抗原主要有血凝素蛋白(Hemagglutinin，HA)、神經氨酸酶(Neuraminidase，NA)、核蛋白(Nuclear Protein，NP)和基質蛋白M(Matrix2)等多肽分子。由于HA、NA高度變異決定著病原體的特殊性，致使每年都有新的病毒株出現(xiàn)，給流感的防控帶來了難題。 目前，對于流感尚無有效的治療手段，而疫苗免疫接種發(fā)揮著重要的作用。流感疫苗經過五十多年的發(fā)展，主要有滅活全病毒疫苗、裂解疫苗、亞單位疫苗、DNA疫苗和減毒活疫苗。由于流感病毒的抗原變異性大，使得疫苗生產每年都需要更換病毒株。當前使用的注射三價滅活疫苗有效，但在保護效果，特別是交叉免疫保護和接種策略上還不理想；亞單位疫苗使用安全，主要針對HA、NA抗原，有一定的免疫保護效果，但是制備周期長，對于季節(jié)性流感來說不太實用，并且保護譜不全面；DNA疫苗起步較晚，主要針對某一保護性抗原成分設計，仍然是保護不全面，另外抗原用量大、特別是潛在基因整合等瓶頸問題：而傳統(tǒng)的減毒活疫苗可以誘導包括細胞免疫、體液免疫和局部免疫在內的較全面的免疫效應，但仍保留一定殘余毒力，存在潛在的“返祖”現(xiàn)象，因此迫切需要發(fā)展新的疫苗研制手段。近年來，反向遺傳技術的誕生為減毒活疫苗的發(fā)展提供了良好的契機。目前，基于RG(reverse genetics，RG)技術的流感減毒活疫苗成為流感疫苗發(fā)展的方向，1999年Neumann等、Hoffmann等學者建立了完全以質粒為基礎的流感病毒反向遺傳技術，為流感減毒活疫苗的研制提供了新的思路。以冷適應減毒病毒株為背景，拯救重配的流感減毒活疫苗株，進而研發(fā)新一代的流感減毒活疫苗是當今流感疫苗研究的熱點。本研究采用反向遺傳技術，通過8質粒流感病毒拯救系統(tǒng)，以冷適應流感病毒株A／Ann Arbor／6／60(H2N2)的6個內部基因為病毒骨架，與2006～2007年流感病毒株A／New Caledonia／20／99(H1N1)的HA和NA基因重排，建立了重配冷適應H1N1亞型流感病毒株拯救體系，為流感減毒活疫苗的研發(fā)開辟了新的思路，同時為流感的安全、有效免疫預防提供研制策略和理論依據。主要研究內容包括： 一、雙向轉錄／表達載體pAD3000的構建 要想得到拯救成功的重配流感病毒株，必須要有一套完善的拯救體系，而在拯救體系中，表達載體起著至關重要的作用。為了使得轉染質粒在拯救系統(tǒng)中能夠高表達，提高其在細胞中的轉錄翻譯水平，我們對載體pHW2000進行了改造，根據轉染所用細胞的類型，選擇用SV40的多聚A(polyA)信號序列取代牛生長激素(BGH)的多聚腺苷酸信號序列，以期獲得高效的轉染效率和病毒拯救效率，為流感減毒活疫苗的制備提供理論基礎。根據相關文獻報道，利用pcDNA3.1(+)質粒為模板，擴增得到了SV40的多聚A(polyA)信號序列，該片段大小為138bp。同時從國外得到的雙向轉錄／表達載體pHW2000，是在pcDNA3.0的基礎上經過一系列的改造而演化而來的，在該載體的基礎上，我們將其升級為雙向轉錄／表達載體pAD3000，通過PCR擴增和測序驗證基因序列是完全正確的，并且利用序列分析軟件對其全序列進行了分析。以上工作為流感病毒8質粒系統(tǒng)轉錄／表達載體的構建奠定了基礎。 二、流感病毒8質粒系統(tǒng)的構建 利用構建好的雙向轉錄／表達載體pAD3000載體系統(tǒng)，我們選擇冷適應、減毒的流感病毒株A／Ann Arbor／6／60的6個內部基因(PB2、PB1、PA、NP、M、NS)作為拯救病毒骨架，人工合成其基因全序列，同時引入PB1-391E，581G，661T，PB2-265S，NP-34G5個氨基酸的突變位點，根據已發(fā)表的序列設計擴增6個基因的特異引物對。將2006～2007年WHO宣布的H1N1亞型流感病毒疫苗株A／New Caledonia／20／99，接種10～11日齡SPF雞胚增殖，通過蔗糖密度梯度離心純化病毒，提取基因組總RNA，反轉錄成cDNA。根據Hoffmann等發(fā)表的通用引物序列擴增HA和NA基因并測序。為獲得準確的5’和3’端序列，在PCR過程中采用高保真聚合酶，且挑選8～10個克隆測序，保證序列的準確性。經序列分析軟件比對，得到拼接出含5’和3’端非編碼區(qū)的8個基因的全長序列。設計帶有BsmBⅠ、BsaⅠ或AarⅠ酶切位點的引物擴增A／Ann Arbor／6／60的6個內部基因片段和A／New Caledonia／20／99的HA和NA基因，分別克隆入pAD3000，并進行了測序驗證，從而構建了8個polⅠ-polⅡ系統(tǒng)的轉錄／表達質粒，分別命名為pMDV-A-PB2，pMDV-A-PB1，pMDV-A-PA，pMDV-A-NP,pMDV-A-M，pMDV-A-NS，pMDV-A-HA，pMDV-A-NA，以期通過同一個載體、轉染后利用細胞中的polⅠ和polⅡRNA聚合酶、實現(xiàn)vRNA和mRNA的轉錄和表達，為冷適應H1N1亞型流感病毒減毒株的構建提供了保證。 三、重配H1N1亞型流感病毒減毒株的制備 1、冷適應流感病毒拯救系統(tǒng)的驗證 用A／New Caledonia／20／99的2個表面基因(HA和NA)或冷適應病毒株A／Ann Arbor／6／60的任意一個內部基因，而其它內部基因來自A／PR／8／34，進行所有組合的基因重排，即8種7+1和1種6+2組合形式，將相應的轉錄／表達質粒組合，共轉染COS-1細胞，均產生了預期組合、有感染性的H1N1亞型流感病毒，表明構建的8個轉錄／表達載體均能有效工作，且冷適應病毒株的6個內部病毒骨架能夠協(xié)同發(fā)揮作用，為進一步篩選和構建重配冷適應流感病毒株奠定了基礎。同時用整合A／PR／8／34的8個基因的重組質粒作陽性對照，也產生了轉染子病毒。在這個過程中，優(yōu)化共轉染體系，在35mmdish上即可產生足以直接用雞胚增殖的子代病毒。 對7+1組合的PB2／PR8、PR8轉染子代病毒及6+2組合的PR8／rMDV-A進行了初步鑒定和部分生物學特性分析。經電鏡觀察，，轉染子病毒的形態(tài)與野生型流感病毒相似。RT-PCR表明引入的氨基酸突變在子代病毒中存在。HA、HI和IFA(indirect immunofluorecent assay，IFA)顯示有重配病毒產生。PR8／rMDV-A的PFU結果表明低溫如33℃更利于重配病毒的繁殖，經過雞胚傳代和MDCK感染實驗，PR8／rMDV-A病毒能在雞胚中高滴度增殖，感染雞胚的能力強，這與提供表面基因的A／PR／8／34病毒株相似，而且對雞胚的毒力弱、低溫利于病毒增殖這又與提供6個內部基因的A／Ann Arbor／6／60相似。以上這些實驗結果證實了冷適應病毒拯救系統(tǒng)的有效性。 2、H1N1流感病毒減毒疫苗株的拯救及鑒定 利用驗證好的冷適應的流感病毒6個質粒系統(tǒng)，作為拯救流感病毒的骨架，與構建好的2006～2007年流感病毒株A／New Caledonia／20／99(H1N1)的HA和NA基因進行重排，8個重組質粒共轉染COS-1細胞，成功得到了重配H1N1亞型流感減毒病毒株，拯救的流感減毒株在第一代雞胚尿囊液中病毒效價為1:512～1:1024，連續(xù)傳4代之后，發(fā)現(xiàn)病毒效價穩(wěn)定，各代之間HA和NA基因的同源性達99.9％以上，經電鏡觀察發(fā)現(xiàn)重配病毒的形態(tài)與野生流感病毒株沒有明顯差別，并對其生物學特性進行了初步的鑒定，深入的研究工作還在進行之中。以上建立的反向遺傳系統(tǒng)重配冷適應H1N1亞型流感病毒株，為流感病毒減毒活疫苗的研制和黏膜免疫機制研究奠定了堅實的基礎。
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392

【參考文獻】

相關期刊論文 前6條

1 陳稚峰,張立國,董婕,吳昆昱,陳愛s

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