禽流感H6N2病毒NP、PA基因的RNAi研究
摘要
禽流感(Avian influenza,AI)是由正黏病毒科A型流感病毒引起的一种急性全身性或呼吸器官性传染病,严重威胁了世界养禽业,常造成巨大的经济损失。更严峻的是禽流感可以感染人类,并且死亡率较高。因此,加强禽流感疾病的防治研究,无论对于养殖业,还是对于人类的健康都具有及其重要的意义。
目前对于禽流感的防治主要采用切断传播途径和疫苗注射等综合防御措施,但由于禽流感病毒的变异率较高,目前所用疫苗的免疫效果并不是很理想,为此迫使全球科研人员都在努力寻求控制禽流感的新方法。
RNAi(RNA interference)即RNAi,是近年来发现的在生物体内普遍存在的一种古老的生物学现象,它可在转录水平、转录后水平和翻译水平上阻断基因的表达。作为一项新兴的生物技术,RNAi有着广泛的应用前景,特别是对于病毒性疾病的防治,是一种非常理想的方法。为了筛选出理想的禽流感病毒干扰序列,本实验针对禽流感H6N2病毒的NP和PA基因,分别设计了两对的siRNA,将siRNA转染到鸡胚和鸡胚成纤维细胞中,通过细胞病变观察、实时荧光RT-PCR和血凝实验检测,结果表明针对NP和PA基因设计的2对siRNA均抑制了病毒的增值。
Avian influenza is the respiratory or systemic infectious disease which is induced by virus from being sticky Section A-type influenza virus, almost all the wild and domestic birds are infected. World Health Organization regulates that the disease must be reported as a national infectious diseases. In 1887, Avian influenza was first reported in domestic birds of Italy. Followed, an outbreak of epidemic throughout the world causing enormous economic losses. Between the beginning of the twentieth century and the late nineteenth century, the disease occurs mainly in Europe, South America, Southeast Asia, some countries in the region and Egypt. Avian influenza as a result of the high mutation rate, in recent years, the number and scale of Avian influenza around the world is expaning and human infection with avian influenza cases have turn up.
Faced with the increasingly serious situation, the researchers are working hard to find a new method for control of influenza. The study found that RNAi is the ideal way to used in the prevention and treatment of influenza.
RNAi(RNA interference) or PTGS by dsRNA homologous target mRNA resulted in degradation. first, dsRNA was cut into about 21~24 nt fragments by the Dicer enzyme(A-type RNA enzymeⅢ), and then lead to mRNA degradation.RNAi as a new technology, with high efficiency, sequence-specific features such as identification, can specifically inhibit gene expression, from the source of virus replication Currently, RNAi has a Bright futureIn virus research
In this paper, H6N2 avian influenza virus was used for RNA interference research and the main content included purification of H6N2 avian influenza virus and determination of virulence, NP, PA gene sequence analysis and RNA interference experiments.
SPF chicken embryo were used for the proliferation of the virus.Toxicity of the virus was tested through Half of the amount of infection and Half lethal dose. the virus titer was detected by hemagglutination assay
Total RNA was extracted from purified virus and sequenced NP, PA gene coding region of virus. According to the results of NP and PA sequencing to determine the target site of siRNA interference to NP and PA genes. two pairs of siRNA to NP, PA gene was designed.
Chicken embryos were transfected with chemically synthesized siRNA and viruses. the effects of siRNA interference were detected by hemagglutination test and real-time RT-PCR. The results show that siRNA designed could inhibit transcription and expression of target gene. It has varying degrees of inhibition on influenza virus in Chicken embryos. Its also have varying degrees of protection for SPF chicken embryo attacked with influenza virus.
SPF chicken embryo fibroblast cells were transfected with Chemical synthesised siRNA, for detection of the specificity and the interference effects of designed siRNA by cytopathic effect, hemagglutination test and real-time RT-PCR. The results show good specificity of siRNA and no effect of inhibition on cells growth. At the same time, the designed siRNA inhibited the target gene transcription and expression, and the replication of influenza virus in chicken embryo fibroblast cells was also inhibited in varying degree.
目前对于禽流感的防治主要采用切断传播途径和疫苗注射等综合防御措施,但由于禽流感病毒的变异率较高,目前所用疫苗的免疫效果并不是很理想,为此迫使全球科研人员都在努力寻求控制禽流感的新方法。
RNAi(RNA interference)即RNAi,是近年来发现的在生物体内普遍存在的一种古老的生物学现象,它可在转录水平、转录后水平和翻译水平上阻断基因的表达。作为一项新兴的生物技术,RNAi有着广泛的应用前景,特别是对于病毒性疾病的防治,是一种非常理想的方法。为了筛选出理想的禽流感病毒干扰序列,本实验针对禽流感H6N2病毒的NP和PA基因,分别设计了两对的siRNA,将siRNA转染到鸡胚和鸡胚成纤维细胞中,通过细胞病变观察、实时荧光RT-PCR和血凝实验检测,结果表明针对NP和PA基因设计的2对siRNA均抑制了病毒的增值。
Avian influenza is the respiratory or systemic infectious disease which is induced by virus from being sticky Section A-type influenza virus, almost all the wild and domestic birds are infected. World Health Organization regulates that the disease must be reported as a national infectious diseases. In 1887, Avian influenza was first reported in domestic birds of Italy. Followed, an outbreak of epidemic throughout the world causing enormous economic losses. Between the beginning of the twentieth century and the late nineteenth century, the disease occurs mainly in Europe, South America, Southeast Asia, some countries in the region and Egypt. Avian influenza as a result of the high mutation rate, in recent years, the number and scale of Avian influenza around the world is expaning and human infection with avian influenza cases have turn up.
Faced with the increasingly serious situation, the researchers are working hard to find a new method for control of influenza. The study found that RNAi is the ideal way to used in the prevention and treatment of influenza.
RNAi(RNA interference) or PTGS by dsRNA homologous target mRNA resulted in degradation. first, dsRNA was cut into about 21~24 nt fragments by the Dicer enzyme(A-type RNA enzymeⅢ), and then lead to mRNA degradation.RNAi as a new technology, with high efficiency, sequence-specific features such as identification, can specifically inhibit gene expression, from the source of virus replication Currently, RNAi has a Bright futureIn virus research
In this paper, H6N2 avian influenza virus was used for RNA interference research and the main content included purification of H6N2 avian influenza virus and determination of virulence, NP, PA gene sequence analysis and RNA interference experiments.
SPF chicken embryo were used for the proliferation of the virus.Toxicity of the virus was tested through Half of the amount of infection and Half lethal dose. the virus titer was detected by hemagglutination assay
Total RNA was extracted from purified virus and sequenced NP, PA gene coding region of virus. According to the results of NP and PA sequencing to determine the target site of siRNA interference to NP and PA genes. two pairs of siRNA to NP, PA gene was designed.
Chicken embryos were transfected with chemically synthesized siRNA and viruses. the effects of siRNA interference were detected by hemagglutination test and real-time RT-PCR. The results show that siRNA designed could inhibit transcription and expression of target gene. It has varying degrees of inhibition on influenza virus in Chicken embryos. Its also have varying degrees of protection for SPF chicken embryo attacked with influenza virus.
SPF chicken embryo fibroblast cells were transfected with Chemical synthesised siRNA, for detection of the specificity and the interference effects of designed siRNA by cytopathic effect, hemagglutination test and real-time RT-PCR. The results show good specificity of siRNA and no effect of inhibition on cells growth. At the same time, the designed siRNA inhibited the target gene transcription and expression, and the replication of influenza virus in chicken embryo fibroblast cells was also inhibited in varying degree.
引文
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[2]傅生芳,独军政,常惠芸,等.禽流感病毒的分子生物学研究进展[J].动物医学进展,2005,26(5):22-24.
[3]周红坡. H5N1亚型禽流感病毒感染性克隆的构建及其RNA干扰的研究[D].武汉:华中农业大学动物科技和动物医学院,2006.
[4]甘孟侯.禽流感第二版[M].北京:中国农业出版社,2004.
[5] Zebedee S L, Lamb R A. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions [J]. J Virol, 1988, 62: 2762-2772.
[6]梁存军,张一,黎晓敏等.禽流感与禽流感病毒的研究进展[J].兽医研究,2007,2:10-12.
[7] Horimoto T, Kawaoka Y. Pandemic Threat Posed by Avian Influenza A Viruses[J]. Clinl Microbiol Rev, 2001, 14.1: 129-149.
[8] Garcia-Sastre A, Egorov A, Matassov D, et al. Influenza A virus lacking the NS 1 gene replicates in interferon-deficient systems [J]. Virology, 1998, 252: 324-330.
[9] Vogel G. Sequence offers clues to deadly flu [J]. Science, 1998, 16, 279(5349): 324.
[10] Luo G, Chung J, Please P. Alteration of the stalk of influenza virus neuraminidase deletions and insertions [J]. Virus Ras, 1993, 29(2): 141-153.
[11]朱建国,陆苹.禽流感病毒核蛋白研究进展[J].中国预防兽医学报,2003,25(1):72-73.
[12] Scholtissek C, Burger H, Kistner O. The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses [J]. Virology, 1985, 147(2): 287-294.
[13]朱建国. A型流感病毒基质蛋白研究进展.动物医学进展[J],2004,25(2):24-26.
[14] Scholtissek C, Ludwig S, Fitch WN. Analysis of influenza A virus nucleoproteins for the assessment of molecular genetic mechanisms leading tonew phylogenetic virus lingages [J]. Arch Virol, 1993, 13f(3=4): 237-250.
[15] Shimizu K, Handa H, Nakada S, et al. Regulation of influenza virus RNA polymerase activity by cellular and viral factors [J]. Nucleic Acids Res, 1994, 22(23): 5047 -5053.
[16] Biswas S K, Boutz P, Nayak D P. Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins [J]. J Virol, 1998, 72(7): 5493-5501.
[17] Mandler J, Muller K, Scholtissek C. Mutants and revertants of an avian influenxa A virus with temperature- sensitive defects in the nucleoprotein and PB2 [J]. J Virology, 1991, 181(2): 512-519.
[18] Medcalf L, Poole E, Elton D. Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein [J] J. Virol, 1999, 73(9): 7349-7356.
[19] Elton D, Medcalf L, Bishop K. Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding [J]. J Virol, 1999: 73(9): 7357-7367.
[20] Qiu Y, Krug R M. The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear edport of mRNAs containing poly(A) [J]. J Virol, 1994, 68(4): 2425-2432.
[21] Qian XY, Alonso-Caplen F, Krug RM, et al. Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA[J]. J Virol. 1994, 68(4): 2433-2441.
[22]曲章义,刘佳,杨力明,等.流感病毒RNA聚合酶的结构和功能研究进展[J].中华结核不fl呼吸杂志,2004,27(4): 261-263.
[23] Ishihama A. A multi2functional enzyme with RNA polymerase and RNase activities: molecular anatomy of influenza virus RNA polymerase[J]. Biochimie, 1996, 78: 1097-1102.
[24] Lee MT, Bishop K, Medcalf L. Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase [J]. Nucleic Acids Res, 2002, 30: 429-438.
[25] Hooker L,Strong R,Adams R. A sensitive, single-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerases: application to kinetic and inhibitor analysis. Nucleic Acids Res,2001, 29: 2691-2698.
[26]李建丽,马仲彬,王泽霖,等.禽流感病毒遗传变异及其分子机制[J].中国动物检疫. 2004,21(6):44-46.
[27]崔尚金.我国禽流感的流行病学调查[D].北京:中国农业科学院研究生院哈尔滨:中国农业科学院哈尔滨兽医研究所,2005.
[28] Kida H, Kawaoka Y, Naeve C W, et al. Antigenic and genetic conservation of H3 influenza in wild ducks [J]. Virology, 1987, 159: 109-119.
[29] Webster R G, Laver W G, Air G M, et al. Molecular mechanisms of variation in influenza viruses [J]. Nature, 1982, 296: 115-121.
[30] Webster RG, Laver W G. The origin of pandemic influenza [J]. Bull W H O, 1982, 47: 449-452.
[31] Bean W J, Cox N J, Kendal AP. Recombinations of human in fluenza A virus in nature [J]. Nature, 1980, 284: 638-640.
[32] Horimoto T, Kawaoka Y. Pandemic Threat Posed by Avian Influenza A Viruses [J]. Clinl Microbiol Rev, 2001, 14.1: 129-149.
[33]李玉清,王宁.流感的分子流行病学研究进展[J].疾病控制杂志. 2001, 5(1): 60-62.
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