双向转录/表达载体的构建及重配H9N1亚型流感病毒的拯救
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摘要
禽流感(Avian Influenza, AI)即禽流行性感冒,是一种烈性传染病,被OIE归为A类传染病。近年来,AI已成为为人类面临的最严重的健康威胁之一。
本研究以pcDNA3质粒为骨架自行构建双向转录/表达载体pHW2008,然后将人工合成的鸡胚高度复制流感病毒株A/Puerto Rico/8/34(H1N1)的8个内部基因cDNA克隆到其上得到8个pol I-pol II系统的转录/表达质粒,分别命名为pHW2008-PB2、pHW2008-PB1、pHW2008-PA、pHW2008-HA、pHW2008-NP、pHW2008-NA、pHW2008-M、pHW2008-NS。接着将这8个重组质粒,转染293T与MDCK混养细胞,利用细饱中的polI和pol II RNA聚合酶,转录出vRNA和mRNA,完成病毒粒子的组装,接着由产生的病毒粒子进入自然感染条件下的增殖过程,从而拯救出病毒,然后经接SPF鸡胚得到大量病毒,并通过电镜确定病毒拯救出来,由此证明此系统的成功建立。
在建立鸡胚高度复制流感病毒株A/Puerto Rico/8/34 (H1N1) 8质粒系统后,提取本室分离的AIV Isolate3(H9N2)基因组,经RT-PCR的方法得到HA基因,将其克隆双向转录/表达载体pHW2008载体上,得到HA转录/表达质粒。接着与构建好的包含A/ Puerto Rico/8/34(H1N1)七个内部基因双向转录/表达质粒pHW2008-PB2、pHW2008-PB1、pHW2008-PA、pHW2008-NP、pHW2008-NA、pHW2008-M、pHW2008-NS,共转染293T与MDCK混养细胞,拯救出重配H9N1亚型流感病毒减毒株,还对其进行了部分生物学特性分析。我们建立的重配H9N1亚型流感病毒株反向遗传系统将为下续工作禽流感病毒变异机理研究及流感减毒活疫苗的研制奠定了坚实的基础。
Avian Influenza (AI) or Avian Influenza, commonly called as Fowl Plague or European roup, is a highly contagious disease of poultry caused by Avian influenza virus which belongs to A-type influenza virus in orthomyxoviridae family.This disease has been classified as A class infectious diseases by OIE and can be divided into highly pathogenic avian influenza, low-pathogenic and non-pathogenic avian influenza according to the nature of pathogen. Infected with the non-pathogenic bird flu, birds have no obvious symptoms ,but can produce antibodies;Infected with the non-pathogenic bird flu, Birds have no obvious symptoms ,but can produce antibodies; Infected with low-pathogenic avian influenza, Birds have mild respiratory symptoms, reduced food intake , drop in egg production and sometimes happen sporadic death ; Infected with the highly pathogenic avian influenza (HPAI),there are a high clinical morbidity and mortality, and often annihilated in infected fl℃ks .Historically, three times of an influenza pandemic brought huge losses to human life and property, of which the most serious is the 1918 -1919 influenza pandemic, resulting in approximately 4000 million to 5000 million people's death in world-wide. Over the past 10 years, the highly pathogenic avian influenza virus have large-scale epidemic in the world, causing the poultry, wild birds, mammals and human infections and deaths, which result in huge losses, and is of great the international community and the World Health Organization ( WHO) concern. But , people mostly concerned about the problem of human infection with highly pathogenic avian influenza viruses at the moment and there are a high mortality rate in people infected by the highly pathogenic avian influenza virus according to the data provided by WHO .Therefore, the control of the bird flu has become more pressing than ever.
The successful establishment of Influenza virus reverse genetics technology provides a powerful tool for the study on the avian flu .The establish of plasmid-based reverse genetics operating system for influenza virus ,especially eight plasmids rescue system constructed by Erich Hoffmann bring a simple, convenient operating platforms for the research of avian flu virus. In this dissertation, we used the eight-plasmid rescue system and selected 7 internal gene of influenza strain A/ Puerto Rico/8/34 (H1N1) as the genetic backbone for generation of recombinant H9N1 subtype influenza virus and 7+1 reassorttants expressing the HA from AIV Isolate3(H9N2) separated by our lab and identified by the Harbin Veterinary Institute, which are well-targeted and very safe for public health , and lay the foundation for the study on the epidemy of influenza viruses, trans-species dissemination and so on .The study include:
1. The construction of bi-directional transcription / expression vector pHW2008
In order to rescue the virus Successful , an appropriate expression vector is necessary, which plays a vital role, and the rescue of influenza virus reassortment strains is not an exception. Because there is no bi-directional transcription / expression vector commercial product , according to reference, we construct the bi-directional transcription / expression vector. Firstly ,we derive the backbone of pHW2008 from the cloning vector pcDNA3. containing the immediate early promoter of the human cytomegalovirus (CMV) and the poly A site of the gene encoding bovine growth hormone (BGH), then insert synthetic human pol I-promoter and terminator sequences between the polIipromoter and the polyA-site.finally identified it by Restriction Endonuclease Reaction and sequence and verified its functions ,which provide condition for the establish of eight Plasmids system.
2.The construction of eight plasmids system for influenza virus
Firstly, We digest 8 internal gene cDNA of influenza strain A/ Puerto Rico/8/34 (H1N1) which are chicken embryo-adapted from these recombinant plasmids: pMD18-T-PB2, pMD18-T-PB1, pMD18-T-PA , pMD18-T-HA, pMD18-T-NP, pMD18-T-NA, pMD18-TM, pMD18-T-NS, then cloned them into the transcription/expression vector pHW2008 vector to produce 8 polI-polII system, the transcription/expression plasmids: pHW2008-PB2,pHW2008-PB1,pHW2008-PA,pHW2008-HA,pHW2008-NP,pHW2008-NA, pHW2008-M, pHW2008-NS,finally, verified them by sequence. After constructed, these eight recombinant plasmids are transfected into 293T /MDCK cells, in which vRNA and mRNA can be transcripted under the use of polI and pol II RNA polymerase, and are assembled into virus particles which can go into the proliferation pr℃ess just like natural infection .After saving the virus, we get a large number of the virus by culture in SPF and verified virus by transmission electron microscopy which proved that this rescue system is established successfully.
3 Rescue of reassortant attenuated H9N1 subtype influenza virus
After the establishment of 8 plasmids rescue system for A / Puerto Rico/8/34 (H1N1), we decided to rescue Reassortant attenuated H9N1 Subtype Influenza Virus. First, we get HA gene by extracted AIV Isolate3 (H9N2) genome and cloned it into pMD18-T vector identified by sequence,then get HA transcription / expression plasmid pHW2008-H9HAby clone the correct HA gene from AIV Isolate3 (H9N2) into bi-directional transcription / expression vector pHW2008 vector.After these,we rescue Reassortant attenuated H9N1 Subtype Influenza Virus by transfection pHW2008-PB2, pHW2008-PB1, pHW2008-PA, pHW2008-NP, pHW2008-NA, pHW2008-M , pHW2008-NS and pHW2008-H9HA into 293T and MDCK cells, and verified it by hemagglutination test, RT-PCR method, Gene chip technology and transmission electron microscope, then, study its biological characterization.The establishment of reverse genetic system for Reassortant attenuated H9N1 Subtype Influenza Virus laid a solid foundation for next works:study on mutation mechanism of bird flu virus and manufacture of live attenuated influenza vaccine .
本研究以pcDNA3质粒为骨架自行构建双向转录/表达载体pHW2008,然后将人工合成的鸡胚高度复制流感病毒株A/Puerto Rico/8/34(H1N1)的8个内部基因cDNA克隆到其上得到8个pol I-pol II系统的转录/表达质粒,分别命名为pHW2008-PB2、pHW2008-PB1、pHW2008-PA、pHW2008-HA、pHW2008-NP、pHW2008-NA、pHW2008-M、pHW2008-NS。接着将这8个重组质粒,转染293T与MDCK混养细胞,利用细饱中的polI和pol II RNA聚合酶,转录出vRNA和mRNA,完成病毒粒子的组装,接着由产生的病毒粒子进入自然感染条件下的增殖过程,从而拯救出病毒,然后经接SPF鸡胚得到大量病毒,并通过电镜确定病毒拯救出来,由此证明此系统的成功建立。
在建立鸡胚高度复制流感病毒株A/Puerto Rico/8/34 (H1N1) 8质粒系统后,提取本室分离的AIV Isolate3(H9N2)基因组,经RT-PCR的方法得到HA基因,将其克隆双向转录/表达载体pHW2008载体上,得到HA转录/表达质粒。接着与构建好的包含A/ Puerto Rico/8/34(H1N1)七个内部基因双向转录/表达质粒pHW2008-PB2、pHW2008-PB1、pHW2008-PA、pHW2008-NP、pHW2008-NA、pHW2008-M、pHW2008-NS,共转染293T与MDCK混养细胞,拯救出重配H9N1亚型流感病毒减毒株,还对其进行了部分生物学特性分析。我们建立的重配H9N1亚型流感病毒株反向遗传系统将为下续工作禽流感病毒变异机理研究及流感减毒活疫苗的研制奠定了坚实的基础。
Avian Influenza (AI) or Avian Influenza, commonly called as Fowl Plague or European roup, is a highly contagious disease of poultry caused by Avian influenza virus which belongs to A-type influenza virus in orthomyxoviridae family.This disease has been classified as A class infectious diseases by OIE and can be divided into highly pathogenic avian influenza, low-pathogenic and non-pathogenic avian influenza according to the nature of pathogen. Infected with the non-pathogenic bird flu, birds have no obvious symptoms ,but can produce antibodies;Infected with the non-pathogenic bird flu, Birds have no obvious symptoms ,but can produce antibodies; Infected with low-pathogenic avian influenza, Birds have mild respiratory symptoms, reduced food intake , drop in egg production and sometimes happen sporadic death ; Infected with the highly pathogenic avian influenza (HPAI),there are a high clinical morbidity and mortality, and often annihilated in infected fl℃ks .Historically, three times of an influenza pandemic brought huge losses to human life and property, of which the most serious is the 1918 -1919 influenza pandemic, resulting in approximately 4000 million to 5000 million people's death in world-wide. Over the past 10 years, the highly pathogenic avian influenza virus have large-scale epidemic in the world, causing the poultry, wild birds, mammals and human infections and deaths, which result in huge losses, and is of great the international community and the World Health Organization ( WHO) concern. But , people mostly concerned about the problem of human infection with highly pathogenic avian influenza viruses at the moment and there are a high mortality rate in people infected by the highly pathogenic avian influenza virus according to the data provided by WHO .Therefore, the control of the bird flu has become more pressing than ever.
The successful establishment of Influenza virus reverse genetics technology provides a powerful tool for the study on the avian flu .The establish of plasmid-based reverse genetics operating system for influenza virus ,especially eight plasmids rescue system constructed by Erich Hoffmann bring a simple, convenient operating platforms for the research of avian flu virus. In this dissertation, we used the eight-plasmid rescue system and selected 7 internal gene of influenza strain A/ Puerto Rico/8/34 (H1N1) as the genetic backbone for generation of recombinant H9N1 subtype influenza virus and 7+1 reassorttants expressing the HA from AIV Isolate3(H9N2) separated by our lab and identified by the Harbin Veterinary Institute, which are well-targeted and very safe for public health , and lay the foundation for the study on the epidemy of influenza viruses, trans-species dissemination and so on .The study include:
1. The construction of bi-directional transcription / expression vector pHW2008
In order to rescue the virus Successful , an appropriate expression vector is necessary, which plays a vital role, and the rescue of influenza virus reassortment strains is not an exception. Because there is no bi-directional transcription / expression vector commercial product , according to reference, we construct the bi-directional transcription / expression vector. Firstly ,we derive the backbone of pHW2008 from the cloning vector pcDNA3. containing the immediate early promoter of the human cytomegalovirus (CMV) and the poly A site of the gene encoding bovine growth hormone (BGH), then insert synthetic human pol I-promoter and terminator sequences between the polIipromoter and the polyA-site.finally identified it by Restriction Endonuclease Reaction and sequence and verified its functions ,which provide condition for the establish of eight Plasmids system.
2.The construction of eight plasmids system for influenza virus
Firstly, We digest 8 internal gene cDNA of influenza strain A/ Puerto Rico/8/34 (H1N1) which are chicken embryo-adapted from these recombinant plasmids: pMD18-T-PB2, pMD18-T-PB1, pMD18-T-PA , pMD18-T-HA, pMD18-T-NP, pMD18-T-NA, pMD18-TM, pMD18-T-NS, then cloned them into the transcription/expression vector pHW2008 vector to produce 8 polI-polII system, the transcription/expression plasmids: pHW2008-PB2,pHW2008-PB1,pHW2008-PA,pHW2008-HA,pHW2008-NP,pHW2008-NA, pHW2008-M, pHW2008-NS,finally, verified them by sequence. After constructed, these eight recombinant plasmids are transfected into 293T /MDCK cells, in which vRNA and mRNA can be transcripted under the use of polI and pol II RNA polymerase, and are assembled into virus particles which can go into the proliferation pr℃ess just like natural infection .After saving the virus, we get a large number of the virus by culture in SPF and verified virus by transmission electron microscopy which proved that this rescue system is established successfully.
3 Rescue of reassortant attenuated H9N1 subtype influenza virus
After the establishment of 8 plasmids rescue system for A / Puerto Rico/8/34 (H1N1), we decided to rescue Reassortant attenuated H9N1 Subtype Influenza Virus. First, we get HA gene by extracted AIV Isolate3 (H9N2) genome and cloned it into pMD18-T vector identified by sequence,then get HA transcription / expression plasmid pHW2008-H9HAby clone the correct HA gene from AIV Isolate3 (H9N2) into bi-directional transcription / expression vector pHW2008 vector.After these,we rescue Reassortant attenuated H9N1 Subtype Influenza Virus by transfection pHW2008-PB2, pHW2008-PB1, pHW2008-PA, pHW2008-NP, pHW2008-NA, pHW2008-M , pHW2008-NS and pHW2008-H9HA into 293T and MDCK cells, and verified it by hemagglutination test, RT-PCR method, Gene chip technology and transmission electron microscope, then, study its biological characterization.The establishment of reverse genetic system for Reassortant attenuated H9N1 Subtype Influenza Virus laid a solid foundation for next works:study on mutation mechanism of bird flu virus and manufacture of live attenuated influenza vaccine .
引文
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