A型流感病毒NS1基因密碼子去優(yōu)化改造引起病毒毒力減弱的研究
發(fā)布時(shí)間：2018-12-11 17:51
【摘要】： 疫苗接種是預(yù)防流感最有效的方法,目前投入使用的流感疫苗可分為滅活苗和減毒活苗兩大類(lèi):滅活苗通過(guò)肌肉注射途徑免疫,能夠誘發(fā)機(jī)體產(chǎn)生針對(duì)血凝素的中和抗體,但無(wú)法刺激鼻腔黏膜產(chǎn)生分泌型抗體,也無(wú)法誘導(dǎo)產(chǎn)生細(xì)胞免疫應(yīng)答;減毒活苗(冷適應(yīng)減毒活苗)通過(guò)鼻吸入的方式免疫,其免疫的方式更接近自然感染的過(guò)程,可充分誘導(dǎo)體液免疫及細(xì)胞免疫應(yīng)答。減毒活苗無(wú)論從免疫途徑還是免疫效果上都優(yōu)于傳統(tǒng)滅活苗。自反向遺傳操作技術(shù)建立以來(lái),研究者們嘗試過(guò)多種方法開(kāi)發(fā)減毒活苗,降低流感病毒毒力,提高其免疫原性,研究出安全有效的減毒活苗已成為新型流感疫苗的發(fā)展方向。 在本研究中,我們根據(jù)A型流感病毒密碼子使用偏嗜性的統(tǒng)計(jì)規(guī)律,在保留NS片段包裝信號(hào)和不影響NS1/NS2剪切位點(diǎn)的前提下,選取稀有密碼子對(duì)A/Puerto Rico/8/34(H1N1)病毒NS1基因內(nèi)部110個(gè)氨基酸區(qū)域進(jìn)行密碼子同義突變改造,將其替換為病毒使用頻率最低的密碼子,全基因合成NS基因,利用12質(zhì)粒反向遺傳操作技術(shù)拯救出含有密碼子去優(yōu)化NS1基因的重組病毒(deoptimized NS, deoNS)。MDCK細(xì)胞噬斑形成實(shí)驗(yàn)表明,病毒在細(xì)胞上的成斑能力大大減弱;病毒在MDCK細(xì)胞上的生長(zhǎng)曲線表明,其在細(xì)胞內(nèi)的復(fù)制能力比野生型病毒低約1000倍。BALB/c小鼠體內(nèi)致病力實(shí)驗(yàn)證明,deoNS病毒不能引起小鼠發(fā)病和死亡,感染第3天病毒在小鼠肺內(nèi)的復(fù)制滴度比野生型病毒低100倍,感染第5天其在小鼠肺內(nèi)的滴度比野生型病毒低1000倍。 本研究初步探索了通過(guò)基因組密碼子去優(yōu)化改造途徑降低A型流感病毒毒力的可行性,首次證明流感病毒NS1基因密碼子去優(yōu)化同義突變可以降低病毒毒力,為流感減毒活疫苗的研究提供了新的思路。
[Abstract]:Vaccination is the most effective way to prevent influenza. The influenza vaccine currently in use can be divided into two categories: inactivated vaccine and live attenuated vaccine: the inactivated vaccine is immunized by intramuscular injection, which can induce the body to produce neutralizing antibodies against hemagglutinin. However, it can not stimulate nasal mucosa to produce secretory antibody, nor can it induce cellular immune response. Attenuated live vaccine (cold acclimated attenuated vaccine) is immunized by nasal inhalation, which is closer to the process of natural infection and can fully induce humoral and cellular immune responses. The live attenuated vaccine is superior to the traditional inactivated vaccine in both the immune pathway and the immune effect. Since the establishment of reverse genetic operation technology, researchers have tried many methods to develop live attenuated vaccine, reduce the virulence of influenza virus, improve its immunogenicity, and find out that the safe and effective live attenuated vaccine has become the development direction of new influenza vaccine. In this study, according to the statistical rule of codon usage bias of influenza A virus, we kept the NS fragment packaging signal and did not affect the NS1/NS2 splicing site. A rare codon was selected to modify the 110 amino acid region of the NS1 gene of A/Puerto Rico/8/34 (H1N1) virus. The codon was replaced by the codon with the lowest frequency of the virus, and the whole gene was synthesized into the NS gene. Using 12 plasmid reverse genetic manipulation technique to save the recombinant virus (deoptimized NS, deoNS). MDCK cell plaque formation with codon to optimize the NS1 gene, the results showed that the ability of the virus to form plaque on the cell was greatly weakened. The growth curve of the virus on MDCK cells showed that the replication ability of the virus in the cells was about 1000 times lower than that of the wild type virus. The virulence test in BALB/c mice showed that deoNS virus could not cause the disease and death of the mice. On the 3rd day of infection, the replication titer of the virus in the lung of mice was 100 times lower than that of the wild type virus, and the titer of the virus in the lung of the mice on the 5th day was 1000 times lower than that of the wild type virus. This study preliminarily explored the feasibility of reducing the virulence of influenza A virus by optimizing the route of genome codon modification. It is the first time that the codon optimization of NS1 gene codon of influenza virus can reduce the virulence of influenza virus. It provides a new idea for the study of live attenuated influenza vaccine.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392
[Abstract]:Vaccination is the most effective way to prevent influenza. The influenza vaccine currently in use can be divided into two categories: inactivated vaccine and live attenuated vaccine: the inactivated vaccine is immunized by intramuscular injection, which can induce the body to produce neutralizing antibodies against hemagglutinin. However, it can not stimulate nasal mucosa to produce secretory antibody, nor can it induce cellular immune response. Attenuated live vaccine (cold acclimated attenuated vaccine) is immunized by nasal inhalation, which is closer to the process of natural infection and can fully induce humoral and cellular immune responses. The live attenuated vaccine is superior to the traditional inactivated vaccine in both the immune pathway and the immune effect. Since the establishment of reverse genetic operation technology, researchers have tried many methods to develop live attenuated vaccine, reduce the virulence of influenza virus, improve its immunogenicity, and find out that the safe and effective live attenuated vaccine has become the development direction of new influenza vaccine. In this study, according to the statistical rule of codon usage bias of influenza A virus, we kept the NS fragment packaging signal and did not affect the NS1/NS2 splicing site. A rare codon was selected to modify the 110 amino acid region of the NS1 gene of A/Puerto Rico/8/34 (H1N1) virus. The codon was replaced by the codon with the lowest frequency of the virus, and the whole gene was synthesized into the NS gene. Using 12 plasmid reverse genetic manipulation technique to save the recombinant virus (deoptimized NS, deoNS). MDCK cell plaque formation with codon to optimize the NS1 gene, the results showed that the ability of the virus to form plaque on the cell was greatly weakened. The growth curve of the virus on MDCK cells showed that the replication ability of the virus in the cells was about 1000 times lower than that of the wild type virus. The virulence test in BALB/c mice showed that deoNS virus could not cause the disease and death of the mice. On the 3rd day of infection, the replication titer of the virus in the lung of mice was 100 times lower than that of the wild type virus, and the titer of the virus in the lung of the mice on the 5th day was 1000 times lower than that of the wild type virus. This study preliminarily explored the feasibility of reducing the virulence of influenza A virus by optimizing the route of genome codon modification. It is the first time that the codon optimization of NS1 gene codon of influenza virus can reduce the virulence of influenza virus. It provides a new idea for the study of live attenuated influenza vaccine.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392
【共引文獻(xiàn)】
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2 陳柳;余斌;云濤;倪征;華炯鋼;李雙茂;;禽類(lèi)病毒免疫逃避的機(jī)制[J];黑龍江畜牧獸醫(yī);2011年11期
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