流感病毒血凝素基因抗流感病毒感染关键序列解析及嵌合基因疫苗的研究

发布时间：2018-06-03 06:32

本文选题：B型流感病毒 + H5N1　；参考：《中南大学》2008年博士论文

【摘要】： (一)B型流感病毒血凝素基因抗同型流感病毒感染关键序列解析血凝素(HA)是B型流感病毒主要的表面糖蛋白。B/Ibaraki/2/85 HA基因全长1758bp,包括信号肽序列、HA1和HA2序列。我们以前的研究表明B/Ibaraki/2/85 HA-DNA在小鼠模型上诱导的免疫反应可以对同型病毒的攻击提供完全保护。本研究通过PCR的方法构建一系列从B/Ibaraki/2/85 HA基因的3'端或5'端连续缺失的裁短HA质粒,用这些质粒免疫BALB/c小鼠,并以致死量的同型病毒攻击小鼠,通过观察小鼠的存活率、体重丢失率、血清中HA抗体的效价和肺部病毒量来寻找HADNA抗流感病毒的关键序列。实验结果表明,HADNA从3'端连续缺失885个核苷酸(295个氨基酸)或5'端信号肽部分连续缺失9个核苷酸,HA-DNA失去保护作用。保留HA的信号肽,HA DNA从5'端连续缺失51个核苷酸(17个氨基酸),HA-DNA失去保护作用。随着HADNA从3'端或5'端连续缺失的核苷酸数目增多,HA-DNA诱导产生的抗体效价下降。B型流感病毒HA DNA的nt94-nt876片段在HA DNA疫苗抗同型流感病毒感染中起重要作用。 (二)A、B型流感病毒嵌合基因疫苗的构建及免疫保护研究本研究在证实A型和B型流感病毒HA1 DNA及关键序列DNA能提供抗同型流感病毒保护的基础上,将编码A型和B型HA1或关键序列基因(A/PR/8/34HA基因nt1-826,B/Ibaraki/2/85 HA基因nt1-876)构建在同一质粒中,制备成嵌合DNA疫苗。将这些重组质粒免疫小鼠,二免后一周,用致死量流感病毒A/PW8/34或B/Ibaraki/2/85攻击,通过测定小鼠血清抗HA抗体和保护效果(包括小鼠的体重丢失率、存活率和肺部病毒量)来评价DNA疫苗的免疫效果。结果表明:A、B型流感病毒HA1基因嵌合疫苗、关键序列基因嵌合疫苗都能抗两种流感病毒致死量的攻击,具有交叉保护的能力。 (三)H5N1型流感病毒血凝素基因抗致死量同型流感病毒感染关键序列解析通过禽流感病毒A/Chicken/JiangSu/01/2002(H5N1)肺对肺感染BALB/c雌性小鼠,多次传代使小鼠致死,建立了小鼠感染模型。以H5N1小鼠适应株HA-DNA免疫小鼠,探讨HA-DNA疫苗和灭活疫苗混合免疫、单独免疫抗同型流感病毒感染的保护效果。结果表明,H5N1 HA-DNA能提供抗致死量同型流感病毒的攻击作用,其保护效果与灭活疫苗无显著差异。在此基础上,我们用PCR的方法构建了一系列裁短质粒,包括3'端连续缺失的裁短质粒和5'端保留信号肽序列的裁短质粒。用这些质粒免疫BALB/c小鼠,并以致死量同型病毒攻击。通过观察小鼠的存活率、体重丢失率、血清中HA抗体的效价和肺部病毒量来寻找H5 HA基因抗流感病毒感染的关键序列。结果显示,HA DNA从3'端连续缺失849个核苷酸(283个氨基酸)或保留HA的信号肽,HA DNA从5'端连续缺失81个核苷酸(27个氨基酸),HA-DNA失去保护作用。H5N1鼠适应株HA基因抗同型流感病毒感染的关键序列为nt127-nt861。
[Abstract]:Analysis of key sequences of hemagglutinin gene of influenza virus type B against influenza virus infection Hemagglutinin (HA) is the main surface glycoprotein. B / Ibaraki / 2 / 85 HA gene of influenza B virus has a total length of 1758bpincluding signal peptide sequences such as HA1 and HA2. Our previous studies have shown that the immune response induced by B/Ibaraki/2/85 HA-DNA in mouse models provides complete protection against the attack of homoviruses. In this study, we constructed a series of truncated HA plasmids continuously deleted from 3'or 5 'end of B/Ibaraki/2/85 HA gene by PCR method. We immunized BALB/c mice with these plasmids and attacked mice with lethal amount of homotypic virus. The survival rate of mice was observed. Body weight loss rate, serum HA antibody titer and lung virus volume were used to search for the key sequence of HADNA anti-influenza virus. The results showed that 885 nucleotides 295 amino acids were deleted from the 3 'terminal of HA-DNA, or 9 nucleotides (HA-DNA) were partially deleted from the 5' terminal signal peptide. The HA DNA, a signal peptide that preserves HA, loses its protective effect by successive deletion of 51 nucleotides (17 amino acids) from the 5 'terminal. With the increase in the number of consecutive deletions of HADNA from the 3 'end or 5' end, the antibody titer induced by HA-DNA decreased. The nt94-nt876 fragment of HA DNA of influenza B influenza virus plays an important role in the HA DNA vaccine against influenza virus infection of the same type. Construction and Immunoprotection of Chimeric Gene Vaccine against Influenza B virus On the basis of confirming that HA1 DNA and DNA of influenza A and B can provide protection against influenza virus of the same type, the same plasmid was constructed by encoding the A and B HA1 or the key sequence gene, nt1-826B / Ibaraki / 2 / 85 HA / nt1-876 / nt1-876, respectively. A chimeric DNA vaccine was prepared. Mice were immunized with these recombinant plasmids. One week after the second immunization, the mice were attacked with lethal influenza virus A/PW8/34 or B/Ibaraki/2/85, and their serum anti-HA antibodies and protective effects (including the weight loss rate of mice) were measured. Survival and lung virus levels) to evaluate the immune effectiveness of the DNA vaccine. The results showed that the HA1 gene chimeric vaccine and the key gene chimeric vaccine of the two influenza viruses could resist the lethal amount of the two influenza viruses and had the ability of cross-protection. (III) Analysis of key sequences of hemagglutinin Gene of Influenza virus H5N1 against lethal dose of the same Influenza virus infection The lung of BALB/c female mice infected by avian influenza virus A / Chicken / Jiang / 01 / 2002 H 5N _ 1 was subcultured for many times and the mice were killed and the model of mouse infection was established. H5N1 mice were immunized with HA-DNA strain to investigate the protective effect of mixed immunization with HA-DNA vaccine and inactivated vaccine against influenza virus infection. The results showed that the H5N1 HA-DNA could provide the anti-attack effect of the same lethal influenza virus, and the protective effect was not significantly different from that of the inactivated vaccine. On this basis, we constructed a series of truncated plasmids using PCR method, including the truncated plasmids with continuous deletion at the 3 'end and the truncated plasmids with the 5' terminal retaining signal peptide sequences. BALB/c mice were immunized with these plasmids and attacked with a lethal dose of the same virus. The key sequences of H5 HA gene against influenza virus infection were found by observing the survival rate, weight loss rate, serum HA antibody titer and lung virus volume of mice. The results showed that 849 nucleotides and 283-amino acids were deleted from the 3'terminal of HA DNA or 81 nucleotides were deleted from the 5'terminal of HA DNA (27 amino acids lost protection of HA-DNA.) the HA gene of the strain adapted to H5N1 mice was resistant to homotypic flow. The key sequence of virus infection is nt127-nt861.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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