本文選題：B型流感病毒 + H5N1��； 參考：《中南大學(xué)》2008年博士論文

【摘要】： (一)B型流感病毒血凝素基因抗同型流感病毒感染關(guān)鍵序列解析 血凝素(HA)是B型流感病毒主要的表面糖蛋白。B/Ibaraki/2/85 HA基因全長1758bp,包括信號肽序列、HA1和HA2序列。我們以前的研究表明B/Ibaraki/2/85 HA-DNA在小鼠模型上誘導(dǎo)的免疫反應(yīng)可以對同型病毒的攻擊提供完全保護。本研究通過PCR的方法構(gòu)建一系列從B/Ibaraki/2/85 HA基因的3'端或5'端連續(xù)缺失的裁短HA質(zhì)粒,用這些質(zhì)粒免疫BALB/c小鼠,并以致死量的同型病毒攻擊小鼠,通過觀察小鼠的存活率、體重丟失率、血清中HA抗體的效價和肺部病毒量來尋找HADNA抗流感病毒的關(guān)鍵序列。實驗結(jié)果表明,HADNA從3'端連續(xù)缺失885個核苷酸(295個氨基酸)或5'端信號肽部分連續(xù)缺失9個核苷酸,HA-DNA失去保護作用。保留HA的信號肽,HA DNA從5'端連續(xù)缺失51個核苷酸(17個氨基酸),HA-DNA失去保護作用。隨著HADNA從3'端或5'端連續(xù)缺失的核苷酸數(shù)目增多,HA-DNA誘導(dǎo)產(chǎn)生的抗體效價下降。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構(gòu)建及免疫保護研究 本研究在證實A型和B型流感病毒HA1 DNA及關(guān)鍵序列DNA能提供抗同型流感病毒保護的基礎(chǔ)上,將編碼A型和B型HA1或關(guān)鍵序列基因(A/PR/8/34HA基因nt1-826,B/Ibaraki/2/85 HA基因nt1-876)構(gòu)建在同一質(zhì)粒中,制備成嵌合DNA疫苗。將這些重組質(zhì)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攻擊,通過測定小鼠血清抗HA抗體和保護效果(包括小鼠的體重丟失率、存活率和肺部病毒量)來評價DNA疫苗的免疫效果。結(jié)果表明:A、B型流感病毒HA1基因嵌合疫苗、關(guān)鍵序列基因嵌合疫苗都能抗兩種流感病毒致死量的攻擊,具有交叉保護的能力。 (三)H5N1型流感病毒血凝素基因抗致死量同型流感病毒感染關(guān)鍵序列解析 通過禽流感病毒A/Chicken/JiangSu/01/2002(H5N1)肺對肺感染BALB/c雌性小鼠,多次傳代使小鼠致死,建立了小鼠感染模型。以H5N1小鼠適應(yīng)株HA-DNA免疫小鼠,探討HA-DNA疫苗和滅活疫苗混合免疫、單獨免疫抗同型流感病毒感染的保護效果。結(jié)果表明,H5N1 HA-DNA能提供抗致死量同型流感病毒的攻擊作用,其保護效果與滅活疫苗無顯著差異。在此基礎(chǔ)上,我們用PCR的方法構(gòu)建了一系列裁短質(zhì)粒,包括3'端連續(xù)缺失的裁短質(zhì)粒和5'端保留信號肽序列的裁短質(zhì)粒。用這些質(zhì)粒免疫BALB/c小鼠,并以致死量同型病毒攻擊。通過觀察小鼠的存活率、體重丟失率、血清中HA抗體的效價和肺部病毒量來尋找H5 HA基因抗流感病毒感染的關(guān)鍵序列。結(jié)果顯示,HA DNA從3'端連續(xù)缺失849個核苷酸(283個氨基酸)或保留HA的信號肽,HA DNA從5'端連續(xù)缺失81個核苷酸(27個氨基酸),HA-DNA失去保護作用。H5N1鼠適應(yīng)株HA基因抗同型流感病毒感染的關(guān)鍵序列為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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392

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