本文選題：流感病毒　切入點：非結(jié)構(gòu)蛋白(NS1)　出處：《南京醫(yī)科大學》2009年碩士論文　論文類型：學位論文

【摘要】：流感病毒屬正粘病毒科,有包膜、分節(jié)段的負單鏈RNA病毒。流感病毒分為甲、乙、丙三型。甲型流感病毒可分為16個HA亞型和9個NA亞型。上個世紀爆發(fā)四次甲型流感大流行,即1918年西班牙流感(H1N1)、1957年亞洲流感(H2N2)、1968年香港流感(H3N2)和1977年重現(xiàn)的H1N1流感。1997年香港爆發(fā)高致病性禽流感(H5N1)感染人。截至2009年5月6日,WHO共報道了423例感染,258例死亡,死亡率高達61.0%。2009年4月墨西哥爆發(fā)甲型H1N1流感,經(jīng)過短暫的時間,全球33個國家正式報告了5728例流感感染病例。這種病毒已被證實經(jīng)由人-人形式傳播。WHO預警級別已升至5級。 流感病毒容易發(fā)生抗原突變和基因重組,即抗原漂移和抗原轉(zhuǎn)換,產(chǎn)生新的病毒株甚至新的亞型。豬、狗等家畜亦可作為流感的貯存宿主,禽流感和人流感病毒基因也可在其體內(nèi)發(fā)生重組。并且由于野生鳥類與家禽的接觸和遷徙,這都加快了流感病毒基因的重組與變異。因此對流感病毒的全球檢測非常重要。流感也是第一個全球監(jiān)控的疾病。但由于流感疫苗的廣泛使用,進行流感監(jiān)測時必須區(qū)分出疫苗免疫動物和病毒感染動物(DIVA)。為此WHO提出和建立了多種DIVA策略,包括哨兵家禽策略、亞單位疫苗策略、異源神經(jīng)氨酸酶策略和非結(jié)構(gòu)蛋白(NS1)策略等。NS1檢測的最大優(yōu)勢是適用于任何一種甲型流感病毒且只需一種檢測方法,而不受病毒抗原漂移的影響。NS1法較其它幾種方法更快速、方便和經(jīng)濟。 流感的最有效控制手段是疫苗接種,對人高致病性禽流感疫苗研究的一個方向是發(fā)現(xiàn)流感通用型疫苗,即尋找病毒保守且與病毒致病性密切相關的蛋白。這類抗原不能阻止病毒感染宿主,但可減輕所有的甲型流感病毒的病情,減低死亡率。這類疫苗對高致病性流感病毒的預防顯得更有意義。在所有甲型流感病毒中,NS1蛋白高度保守,并與流感病毒致病性密切相關。目前關于NS1蛋白免疫保護作用研究的報道不多且結(jié)論也不相一致。 本試驗主要研究內(nèi)容和結(jié)果如下: 一、流感病毒NS1蛋白在大腸桿菌中的表達、純化及多克隆抗體制備設計NS1特異性引物,克隆NS1全長序列,構(gòu)建重組原核表達質(zhì)粒pET28a-NS1。重組質(zhì)粒經(jīng)PCR、酶切鑒定、核酸序列測定和分析后轉(zhuǎn)化大腸桿菌BL21,經(jīng)誘導表達的融合蛋白用鎳柱親和層析純化,SDS-PAGE和Western blot結(jié)果顯示表達的蛋白分子量約為26 kDa。NS1免疫新西蘭兔后可獲得高效價多克隆抗體(105以上),并可用于Western blot檢測。 二、建立NS1-ELISA檢測方法 以純化NS1蛋白包被96孔板,建立檢測小鼠血清NS1抗體的間接ELISA方法,在病毒感染后15-30d可正確區(qū)分出病毒感染動物和疫苗免疫動物。此方法具有較高的敏感性(94.4%)和特異性(88.9%)。 三、NS1蛋白免疫保護作用研究 NS1純化蛋白兩次免疫小鼠后,第30d小鼠抗體陽性率為84.6%。NS1免疫小鼠的生存率為37.5%,高于病毒對照組(16.0%),但遠低于疫苗免疫組(81.3%)。半數(shù)生存時間、肺病變和肺指數(shù)等指標較病毒對照組沒有顯著差異,說明NS1蛋白不是流感的主要保護性蛋白,NS1蛋白對小鼠免疫保護作用較差。綜上所述,本研究成功克隆了流感病毒NS1基因并在大腸桿菌中表達,并將 此蛋白初步運用于病毒感染小鼠和疫苗免疫小鼠的血清學鑒別檢測。NS1蛋白對小鼠的免疫保護作用較差。本實驗制備的高效價NS1多克隆抗體可用于Western blot檢測,為今后研究NS1的生物學活性奠定了基礎。
[Abstract]:Influenza virus family Orthomyxoviridae envelope, segmented negative strand RNA virus. Influenza viruses are divided into a, B, C three. Influenza A virus can be divided into 16 subtypes of HA and 9 NA subtypes. The last century the outbreak of the four influenza pandemic, the 1918 Spanish flu (i.e. H1N1), the 1957 Asian flu (H2N2), influenza A (H3N2) in Hongkong in 1968 and 1977 to reproduce the H1N1 influenza.1997 in Hongkong outbreak of highly pathogenic avian influenza (H5N1) infection. As of May 6, 2009, WHO reported 423 cases of infection, 258 cases died, the mortality rate as high as 61.0%.2009 in April Mexico outbreak of influenza a H1N1. After a short time, the 33 countries in the world have officially reported 5728 cases of influenza infection. The virus has been confirmed by the people who form the spread.WHO warning level has risen to 5.
The influenza virus antigen prone to mutation and gene recombination, namely antigenic drift or antigenic shift, produce new strains and new subtype. Pigs, dogs and other animals can be used as a reservoir of influenza, avian flu and human influenza virus gene recombination can also occur in its body. And by the Yu Yesheng birds and poultry contact and this migration, are speeding up the reorganization and variation of influenza virus gene. So the detection of global influenza virus is very important. The flu is also the first global monitoring of disease. But due to the widespread use of influenza vaccine, influenza surveillance must distinguish vaccinated animal and animal virus infection (DIVA). Therefore WHO is proposed in this paper a variety of DIVA strategy, including the sentinel poultry subunit vaccine strategy, strategy, strategy and non structural protein heterologous neuraminidase (NS1) the biggest advantage strategy of.NS1 detection is applicable to any Influenza A virus and only one detection method, without the effect of virus antigen drift,.NS1 method is more rapid, convenient and economical than the other methods.
The most effective means of control of influenza vaccine, a direction of highly pathogenic avian influenza vaccine research is to find a universal flu vaccine for the virus, namely conservative and virus pathogenicity related proteins. These antigens can prevent virus infection, but can reduce all influenza A virus disease reduce mortality., to prevent this kind of vaccine against highly pathogenic influenza virus is more important. In all influenza A virus, NS1 protein is highly conserved, and the pathogenicity of influenza virus NS1 protein are closely related. The immune protective effects of the report is not much and the conclusions are not consistent.
The main contents and results of this experiment are as follows:
A, expression of influenza virus NS1 protein in Escherichia coli, purification and preparation of polyclonal antibody for NS1 specific primer, the full-length NS1 clone, recombinant expression plasmid of recombinant pET28a-NS1. plasmid by PCR, enzyme digestion, DNA sequencing and analysis after transformed into Escherichia coli BL21, the inducible expression of the fusion protein by nickel column affinity chromatography, SDS-PAGE and Western blot showed that the molecular weight of the expressed protein was about 26 kDa.NS1 New Zealand rabbits were immunized after high titer polyclonal antibody (more than 105), and can be used for Western blot detection.
Two, the establishment of NS1-ELISA detection method
The purified NS1 protein was coated with 96 porous plates, and an indirect ELISA method was established to detect NS1 antibody in mice serum. After virus infection, 15-30d can correctly distinguish virus infected animals and vaccine immunized animals. This method has high sensitivity (94.4%) and specificity (88.9%).
Three, study on the protective effect of NS1 protein
The NS1 protein was purified two times after immunization, the positive rate of 30d antibody in mice 84.6%.NS1 mice survival rate was 37.5%, higher than that of the virus control group (16%), but much lower than vaccine group (81.3%). The median survival time, there is no significant difference between lung disease and lung index compared with the virus control group, the main protective protein NS1 protein is not the flu, poor NS1 protein on mice immune. In summary, this study successfully cloned the NS1 gene of influenza virus and its expression in Escherichia coli, and
The initial use of this protein to detect.NS1 protein in serum is poor in mice infected with the virus and vaccine immune protection to mice. The experimental preparation of high titer NS1 polyclonal antibody can be used for Western blot detection, which laid the foundation for the future research on biological activity of NS1.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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本文編號：1635559