摘要
MicroRNA (miRNA)是一类长度约22个核苷酸的内源性非编码的小RNA分子,广泛存在于各种生物体内。miRNA通过作用于靶mRNA的3′UTR区,引起靶mRNA的降解或抑制其翻译,从而实现对靶基因表达水平的调控,研究发现,miRNA在细胞发育分化,组织器官形成以及病毒感染等多种生物学过程中均发挥重要作用。
禽流感是由正粘病毒科流感病毒属A型流感病毒引起的以禽类为主的可导致人畜共患的烈性传染性。随着高致病性禽流感病毒感染水禽导致发病死亡的事实出现后,人们越来越意识到禽流感病毒对禽类乃至于对人类的危害。高通量测序技术是近年来发展起来的用于各物种miRNA分析鉴定的强有力工具。鸭miRNA的研究目前还处于空白的阶段,尤其对于感染禽流感病毒后miRNA的变化在水禽中还没有报道,因此本研究在对3月龄SPF鸭感染H5N1亚型禽流感病毒的基础上,采集感染鸭和SPF鸭的脾脏组织,利用高通量测序分析技术,鉴定感染与非感染H5N1亚型禽流感病毒状态下鸭脾脏组织的miRNA并对其进行差异表达分析,同时由于miRNA具有一定的组织特异性,这一特性使得miRNA对生物体的不同生长发育过程及不同的组织器官发挥多种不同的作用。因此本研究进一步对部分差异表达miRNA,利用荧光定量PCR的方法分析其在感染与非感染H5N1亚型禽流感病毒的鸭不同组织中的表达情况。
结果表明,SPF鸭感染H5N1亚型禽流感病毒后的第7天,可以检测到鸭体内病毒的表达,并在第15天达到最大值。利用高通量测序技术,成功获得感染与非感染鸭脾脏组织小RNA文库,总体分析显示,SPF鸭脾脏组织的小RNA种类和数量明显多于感染禽流感病毒鸭的脾脏组织。同时获得了322个差异表达的miRNA,其中105个显著上调,217个显著下调miRNA。另外在新发现的17个小RNA中,有1个显著上调,4个显著下调。随机选取9个差异表达miRNA进行定量分析,检测结果与高通量测序所得结果相一致,证明了高通量测序结果的准确性。不同组织定量分析结果显示,在H5N1亚型禽流感病毒感染状态下,miR-17a*,miR-30c*和miR-10在肌肉中的相对表达量最高,miR-4006f-5p和miR-6在气管中的相对表达量最高,miR-92b-3p在肺脏中的相对表达量最高,miR-4499在脾脏中的相对表达量最高,miR-2808e和miR-4002-5p在心脏中的相对表达量最高。而miR-17a*,miR-92b-3p,miR-30c*,miR-10和miR-6均在肝脏中的相对表达量最低,另外miR-4006f-5p,miR-4499和miR-2808e在肺脏中几乎不表达。在SPF鸭中miR-17a*,miR-30c*和miR-10在肺脏中的相对表达量最高,miR-4006f-5p在肌肉中相对表达量最高,miR-92b-3p和miR-4499在脾脏中的相对表达量最高,miR-6在肾脏中的相对表达量最高,miR-2808e和miR-4002-5p在心脏中的相对表达量最高。而miR-17a*,miR-4006f-5p,miR-10,miR-6和miR-4499均在肝脏中的相对表达量最低。所研究miRNA在病毒感染前后均在不同组织中具有不同的表达量。
本研究对鸭感染与非感染H5N1亚型禽流感病毒状态下的脾脏组织间miRNA组学差异进行了研究,结果表明miRNA在禽流感病毒作用下的水禽中可能具有重要调控作用,该结果为进一步研究miRNA对水禽感染禽流感病毒下的作用机制研究奠定了基础,同时为抗病育种的发展提供新的理论依据。
MicroRNA is a non-coding small RNA with about22nucleotides in lengthendogenous that widely present in a variety of organisms. MiRNA complementarypairing with the target mRNA3′UTR region, specificity base caused the degradationof the target mRNA or inhibiting their translation in order to achieve the level oftarget gene expression regulation. It may paly important role in a variety of biologicalprocesses such as cell development, differentiation, tissues and organa formation,viral infection, et al.
Avian influenza belongs to the orthomyxovirus virus family influenza virus, is apetent infectious poultry, caused by influenza A virus which can lead to human andanimal comorbid. The fact of that the incidence of death with highly pathogenic avianinfluenza virus infection waterfowl appears, human are increasingly aware of hazardsof AIV. High-throughput sequencing technology as a powerful tool for miRNAanalysis identified. Now ducks miRNA research is still in a blank stage, eapeciallyinfected and non-infected AIV duck.
In this study, on the basis of collected3-month-old SPF ducks spleentissueswhich infected and non-infected H5N1subtype avian influenza virus, usinghigh-throughput sequencing analysis techniques, identification and differentiallyexpressed analysis of miRNA in infected and non-infected with the H5N1subtype ofavian influenza virus ducks spleen tissue. The miRNA has a feature of tissue-specific,it makes miRNA play a variety of different role in the process of growth anddevelopment of organisms and different tissues and organs. Further, using quantitativePCR, we analyzed the expression of some differentially expressed miRNAs in ducksvarious tissue under infected and non-infected with the H5N1subtype of avian influenza virus.
The results show that we can detect the avian influenza virus expression in duckvivo after infected avian influenza virus seven days, and reaches a maximum at15days. Deep sequencing of the two libraries by using the high-throughput sequencingtechnology successfully generated. The result shown that the small RNA aresignificant more in types and quantity in SPF duck than the duck that infected H5N1subtype of avian influenza virus.322known differentially expressed miRNAs wereidentified, of which105were significantly up-regulated and217were significantlydown-regulated. In addition,17novel small RNA were identified, including1wassignificantly up-regulated miRNA and4were significantly down-regulated miRNA.The quantitative analysis of nine differentially expressed miRNA that randomlyselected, the result verified the accuracy of the results of the high-throughputsequencing.
The different tissues Quantitative analysis results show that the ducks under theH5N1subtype of avian influenza virus infection, miR-17a*, miR-30c*and miR-10have highest relative expression level in muscle, miR-4006f-5p and miR-6havehighest relative expression level in trachea, miR-92b-3p has highest relativeexpression level in lungs, miR-4499highest relative expression level in spleen,miR-2808e and miR-4002-5p have highest relative expression level in heart.However,miR-17a*, miR-92b-3p, miR-30c*, miR-10and miR-6have lowest relativeexpression lever in liver. In addition, miR-4006f-5p, miR-4499and miR-2808e almosthave no relative expression in lung. In SPF ducks, miR-17a*, miR-30c*, miR-10havehighest relative expression level in lung, miR-4006f-5p has highest relative expressionlevel in muscle, miR-92b-3p and miR-4499have highest relative expression level inspleen, miR-6has highest relative expression level in kidney, miR-2808e andmiR-4002-5p have highest relative expression level in heart. However, miR-17a*,miR-4006f-5p, miR-10, miR-6and miR-4499in liver have lowest relative expressionlevel. In this study, we found that miRNA has a different expression of the same tissue,in SPF duck and infected ducks.
In this study, the miRNA genomics analysis between Duck spleen tissue infectedand non-infected with the H5N1subtype of avian influenza virus were performed, theresults show that miRNA may play an important regulation role in waterfowl in therole of avian influenza virus. This result may lay the foundation for the further studyin miRNA research on the machanism of waterfowl under infected with avianinfluenza virus. At the same time provide a new theoretical basis for the developmentof disease-resistant breeding.
引文
[1] Guarnieri DJ, Dileone RJ. MicroRNAs: a new class of gene regulators[J]. AnnMed,2008,40(3):197-208.
[2] Lee RC, Feinbaum RL, Ambros V. The C.elegans heterochronic gene lin-4encodes small RNAs with antisense complementarity to lin-14[J]. Cell,1993,75(5):843-854.
[3] Reinhart BJ, Slack FJ, Basson M, et al. The21-nucleotide let-7RNA regulatesdevelopmental timing in Caenorhabditis elegans[J]. Nature,2000,403(6772):901-906.
[4] Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence andtemporal expression of let-7heterchronic regulatory RNA[J].Nature,2000,408(6808):86-89.
[5] Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genescoding for small expressed RNAs[J]. Science,2001,294(5543):853-858.
[6] Lau NC, Lim LP, Weinstein EG, et al. An abundant class of tiny RNAs withprobable regulatory roles in Caenorhabditis elegans[J]. Science,2001,294(5543):858-862.
[7] Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans[J]. Science,2001,294(5543):862-864.
[8] Rodriquez A, Griffiths-Jone S, Ashurst JL, et al. Identification of mammalianmicroRNA host genes and transcription units[J]. Genome Res,2004,14(10A):1902-1910.
[9] Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineagedifferentiation [J]. Science,2004,303(5654):83-86.
[10] Zeng Y, Wagner EJ, Cullen BR. Both natural and designed microRNAs caninhibit the expression of cognate mRNAs when expressed in human cells[J]. MolCell,2002,9(6):1327-1333.
[11] Ohler U, Yekta S, Lim LP, et al. Patterns of flanking sequence conservation and acharacteristic upstream motif for microRNA gene identification[J]. RNA,2004,10(9):1309-1322.
[12] Johnston RJ, Hobert O. A microRNA contolling left/right neuronal asymmetry inCaenorhabditis elegans[J]. Nature,2003,426(6968):845-849.
[13] Bracht J, Hunter S, Eachus R, et al. Trans-splicing and polyadenylation of let-7microRNA primary transcripts[J]. RNA,2004,10(10):1586-1594.
[14] Denli AM, Tops BB, Plasterk RH, et al. Processing of primary microRNAs by theMicroprocessor complex[J]. Nature,2004,432(7014):231-235.
[15] Hutv gner G, Zamore PD. A microRNA in a multiple-turnover RNAi enzymecomplex[J]. Science,2002,297(5589):2056-2060.
[16] Lai EC. MicroRNAs are complementary to3'UTR sequence motifs that mediatenegative post-transcriptional regulation[J]. Nat Genet,2002,30(4):363-364.
[17] Tang G, Reinhart BJ, Bartel DP, et al. A biochemical framework for RNAsilencing in plants[J]. Genes Dev,2003,17(1):49-63.
[18] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell,2004,116(2):281-297.
[19] Petersen CP, Bordeleau ME, Pelletier J, et al. Short RNAs repress translationafter initiation in mammalian cells[J]. Mol Cell,2006,21(4):533-542.
[20] Poy MN, Eliasson L, Krutzfeldt J, et al. A pancreatic islet-specific microRNAregulates insulin secretion[J]. Nature,2004,432(7014):226-230.
[21] Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression inzebrafish embryonic development[J]. Science,2005,309(5732):310-311.
[22] Mansfield JH, Harfe BD, Nissen R, et al. MicroRNA-responsive 'sensor'transgenes uncover Hox-like and other developmentally regulated patterns ofvertebrate microRNA expression[J]. Nat Genet,2004.36(10):1079-1083.
[23]Yang WJ, Yang DD, Na S, et al. Dicer is required for embryonic angiogenesisduring mouse development[J]. J Biol Chem,2005,280(10):9330-9335.
[24] Clop A, Marcq F, Takeda H, et al. A mutation creating a potential illegitimatemicroRNA target site in the myostatin gene affects muscularity in sheep[J]. NatGenet,2006,38(7):813-818.
[25] Ivey KN, Muth A, Arnold J, et al. MicroRNA regulation of cell lineages in mouseand human embtyonic stem cells[J]. Cell Stem,2008,2(10):219-229.
[26] Barroso-del Jesus A, Lucena-Aguilar G, Menendez P. The miR-302-367cluster asa potential stemness regulator in ESCs[J]. Cell Cycle,2009,8(3):394-398.
[27] Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineagedifferentiation [J]. Science,2004,303(5654):83-86.
[28] Poy MN, Eliasson L, Krutafeldt J, et al. A pancreatic islet-specific microRNAregulates insulin secretion [J]. Nature,2004,432(7014):226-230.
[29] Xu P, Vernooy SY, Guo M, et al. The Drosophila MicroRNA mir-14suppressescell death and is required for normal fat metabolism [J]. Curr Biol,2003,13(9):790-795.
[30] Kawasaki H, Taira K. Hes1is a target of microRNA-23during retinoic-acid-induced neuronal differentiation of NT2cells[J]. Nature,2003,423(6942):838-842.
[31] Montenegro D, Romero R, Kim SS, et al. Expression patterns of microRNAs inthe chorioamniotic membranes: a role for microRNAs in human pregnancy andparturition[J]. J Pathol2009.217(1):113-121.
[32] Mouillet JF,Chu T, Hubel CA, et al. The levels of hypoxia-regulated microRNAsin plasma of pregnant women with fetal growth restriction[J]. Placenta,2010,31(9):781-784.
[33] Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA gene miR15and miR16at13q14in chroniclympocytic leukemia[J]. Proc Natl Acad Sci U S A,2002,99(24):15524-15529.
[34] Cimmino A, Calin GA, Fabbri M, et al. miR-15and miR-16induce apoptosis bytargeting BCL2[J]. Proc Natl Acad Sci U S A,2005,102(39):13944-13949.
[35] Lorio MV, Visone R, Dileva G, et al. MicroRNA signatures in human ovariancancer[J]. Cancer Res,2007,67(18):8699-8707.
[36] Le Sage C, Nagel R, Egan DA, et al. Regulation of the p27(Kip1) tumorsuppressor by miR-221and miR-222promotes cancer cell proliferation[J].EMBO J,2007,26(15):3699-3708.
[37] Ganofalo M, Quintavalle C, Di Leva G, et al. MicroRNA signatures of TRALLresistance in human non-small cell lung cancer[J]. Oncogene,2008,27(27):3845-3855.
[38]王玫,李思光,罗玉萍. microRNA识别和鉴定方法[J].细胞生物学杂志,2007,29:503-507.
[39] Lim LP, Lau NC, Weinstein EG, et al. The microRNAs of Caenorhabditiselegans[J]. Genes Dev,2003,17(8):991-1008.
[40] Grad Y, Aach J, Hayes GD, et al. Computational and experimental identificationof C.elegans microRNAs[J]. Mol Cell,2003,11(5):1253-1263.
[41] Park W, Li J, Song R, et al. CARPEL FACTORY, a Dicer homolog, and HEN1, anovel protein, act in microRNA metabolism in Arabidopsis thaliana[J]. Curr Biol,2002.12(17):1484-1495.
[42] Lewis BP, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalianmicroRNA targets[J]. Cell,2003,115(7):787-798.
[43] Xie X, Lu J, Kulbokas EJ, et al. Systematic discovery of regulatory motifs inhuman promoters and3' UTRs by comparison of several mammals[J]. Nature2005,434(7031):338-345.
[44] Adai A, Johnson C, Mlotshwa S, et al. Computational prediction of miRNAs inArabidopsis thaliana[J]. Genome Res,2005,15(1):78-91
[45] Liu CG, Calin GA, Meloon B, et al.An oligonucleotide microchip forgenome-wide microRNA profiling in human and mouse tissues [J]. Proc NatlAcad Sci U S A,2004,101(26):9740-9744.
[46] Krichevsky AM, King KS, Donahue CP, et al. A microRNA array revealsextensive regulation of microRNAs during brain development [J]. RNA,2003,9(10):1274-1281.
[47]杨桢. microRNA相关的生物信息学与进化分析[D].上海:复旦大学,2011.
[48] Zhang W, Dahlberg JE, Tam W. MicroRNAs in tumorigenesis: a primer[J]. Am JPathol,2007,171(3):728-738.
[49] Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression inzebrafish embryonic development[J]. Science,2005,309(5732):310-311.
[50] Thompson RC, Deo M, Turner DL. Analysis of microRNA expression by in situhybridization with RNA oligonucleotide probes[J]. Methods,2007,43(2):153-161.
[51] Kloosterman WP, Wienholds E, de Bruijn E, et al. In situ detection of miRNAs inanimal embryos using LNA-modified oligonucteotide probes [J]. Nat Methods,2006,3(1):27-29.
[52] Darnell DK, Kaur S, Stanislaw S, et al. MicroRNA expression during chickembryo development [J]. Dev Dyn,2006,235(1):3156-3165.
[53] Varkonyi-Gasic E, Wu R, Wood M, et al. Protocol: a highly sensitive RT-PCRmethod for detection and quantification of microRNAs[J]. Plant Methods,2007,3:12.
[54] John B, Enright AJ, Aravin A, et al. Human microRNA targets[J]. PLOS Biol,2004,2(11):e363
[55] Baek D, Villen J, Shin C, et al. The impact of microRNAs on protein output[J].Nature,2008,455(7209):64-71.
[56] Selbach M, Schwanhausser B, Thierfelder N, et al. Widespread changes inprotein synthesis induced by microRNAs[J]. Nature,2008455(7209):58-63.
[57] Kiriakidou M, Nelson PT, Kouranov A, et al. A combined computational-experimental approach predicts human microRNA targets[J]. Genes Dev,2004,18(10):1165-1178.
[58]吴红专,刘福安.禽流感与人类健康[J].养禽与禽病防治,1998(5):5-6.
[59]马秀丽,崔言顺.国外禽流感研究进展[J].畜牧市场,2009(2):24-25.
[60] David L, Suarez. Evolution of avian influenza viruses[J]. Vet microbial,2000,74:15-27.
[61]庞坤.禽流感研究进展[J].信阳农业高等专科学校学报,2004,14(1):36-37.
[62]于康震,陈化兰,唐秀英.97香港禽流感[J].中国畜禽传染病,1998(3):189.
[63]黄瑞,李嫄源.禽流感研究进展[J].国外医学(微生物学分册),2005,28(6):31-32.
[64]吴明华,杨永钦,李维召.禽流感流行病学和免疫防制的新进展[J].中国畜牧兽医,2006,33(10):18-21.
[65]张哲强,秦曦明,梁荣等.禽流感病毒HA基因真核表达质粒的构建和表迭[J].生物化学与生物物理进展.2003,30(3):483-487.
[66]乔传玲,于康震.禽流感病毒结构置白及其免疫原性[J].预防兽医学进展,2000,2(4):4-7.
[67]朱建国,陆萍.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.
[68]王幼明,邓国华,于康震,等.禽流感病毒GD/I/96株M基因在sf9昆虫细胞中的表达[J].中国兽医科技,2C02,32(9):3-5.
[69]郭元吉.流行性感冒.见:黄祯祥主编.医学病毒学基础及实验技术[J].北京:北京科技出版社,1990,661-693.
[70]黄平.流感病毒A/Guangdong/28/94HAI基因序列分析[J].中华微生物学和免疫学杂志,1996,16(2):133-134.
[71] Guo YJ Characterization of a new anian-like influenza A virus from horses inChina[J]. Virology,1992(188):245-246.
[72] Verhoeyen M, Fang R, Jou WM, et al. Antigenic drift between thehaemagglutinin of the Hong Kong influenza strains A/Aichi/2/68and A/Victoria/3/75[J]. Nature,1980,286(5775):771-776.
[73] Webster R G, Laver WG, Air GM, et al. The mechanism of antigenic drift ininfluenza viruses: analysis of HongKong (H3N2) variants with monoclonalantibodies to the hemagglutinin molecule[J]. Ann N Y Acad Sci,1980,354:142-161.
[74]王秀荣,陈化兰.禽流感与公共卫生[J].基础兽医学与临床,2005,25(12):1079-1083.
[75]方立群,曹春香,陈国胜.地理信息系统应用于中国大陆高致病性禽流感的空间分布及环境因素分析[J].中华流行病学杂志,2005,11(26):839-842.
[76] Kandun IN,Wibisono H,Sedyaningsih E R,et al. Three Indonesian clusters ofH5N1virus infection in2005[J]. N Engl J Med,2006,355(21):2186-2194.
[77]寇铮,唐利军,雷富民,等.一株H5N1亚型禽流感病毒的分离与鉴定[J].中国病毒学,2004,19(5):490-492.
[78]雷彩侠.中国禽流感诊断技术和疫苗的研究进展[J].中国兽药,2006,40(1):32-39.
[79]李海燕,于康震,辛晓光,等.禽流感间接ELISA诊断试剂盒的研制及医用[J].中国预防兽医学报,2001,23(5):372-376。
[80]王兆鹏,尚绪增,刘思莹,等.禽流感病毒M1抗体间接阻断ELISA检测方法的建立[J].中国兽医科学,2009,39(05):426-431.
[81]李娜,张保平.双抗体夹心ELISA检测禽流感病毒方法的初步建立[J].湖南文理学院学报,2008,20(02):60-62.
[82] Yang M, Clavijo A, Graham J, et al. Production and diagnostic application ofmonoalonal antibodies against influenza virus H5[J]. J Virol Methods,2009,162(1-2):194-202.
[83]李海燕,辛晓光,田国斌,等.禽流感抗体斑点-ELISA诊断技术的研究[J].中国预防兽医学报,1999,21(5):321-324.
[84] Velumani S, Du Q, Fenner, et al. Development of an antigen-capture ELISA fordetection of H7subtype avian influenza from experimentally infectedchickens[J]. J Virol Methods,2008,147(2):219-225.
[85] Watcharatanyatip K, Boonmoh S, Chaichoun K, et al. Multispecies detection ofantibodies to influenza A viruses by a double-antigen sandwich ELISA[J]. J VirolMethods,2010,163(2):238-243.
[86] Van Deusen RA, Hinshaw VS, Senne DA, et al. Micro neuraminidase-inhibitionassay for classification of Influenza A virus neuraminidase[J]. Avian Dis,1983,27(3):745-750.
[87]张应国,宋建领,胡媛媛,等.禽流感病毒RT-PCR及多重RT-PCR检测技术的建立[J].中国兽医科技,2005,35(8):600-604.
[88] Xie Z, Pang YS, Liu J, et al. A multiplex RT-PCR for detection of type Ainfluenza virus and differentiation of avain H5, H7, and H9hemagglutininsubtypes[J]. Mol Cell Probes,2006,20(3-4):245-249.
[89] Chen HT, Zhang J, Ma LN, et al. Rapid subtyping of H9N2influenza virus by atriple reverse transcription polymerase chain reaction[J]. J Virol Methods,2009,158(1-2):58-62.
[90] Yacoub A, Kiss I, Zohari S, et al. The rapid molecular subtyping and pathotypingof avain influenza viruses[J]. J Virol Methods,2009,156(1-2):157-161.
[91] Lee CW, Suarez DL. Application of real-time RT-PCR for the quantitation andcompetitive replication study of H5and H7subtype avian influenza virus[J]. JVirol Methods,2004,119(2):151-158.
[92] Stone B, Burrows J, Schepetiuk S,et al. Rapid detection and simultaneoussubtype differentiation of influenza A viruses by real time PCR[J]. J VirolMethods,2004,117(2):103-112.
[93] Cattoli G, Drago A, Maniero S, et al. Comparison of three rapid detectionsystems for type A influenza virus on tracheal swabs of experimentally andnaturally infected birds[J]. Avian Pathol,2004,33(4):432-437.
[94] Muradrasoli S, Mohamed N, Belák S, et al. Broadly targeted triplex real-timePCR detection of influenza A, B and C viruses based on the nucleoprotein geneand a novel “MegaBeacon” probe strategy[J]. J Virol Methods,2010,163(2):313-322.
[95]卢亦愚,严菊英,冯燕,等. TaqMan-MGB荧光定量RT-PCR技术快速检测[J].中国病毒学,2006,21(5):472-476.
[96]刘丽玲,蒋永萍,王秀荣,等. SYBR Green I荧光RT-PCR方法检测禽流感病毒[J].畜牧兽医学报,2006,37(11):1189-1193.
[97] Lau LT, Banks J, Aherne R, et al. Nucleic acid sequence-based amplificationmethods to detect avain influenza virus[J].Biochem Biophys Res Commun,2004,313(2):336-342.
[98]黄庚明,辛朝安. PCR制备地高辛标记的探针检测禽流感病毒核酸[J].中国兽医杂志,2001,37(12):3-7.
[99] Chen HT, Zhang J, Sun DH, et al. Development of reverse transcriptionloop-mediated isothermal amplification for rapid detection of H9avain influenzavirus[J]. J Virol Methods,2008,151(2):200-203.
[100] Fukuta S, Ohishi K, Yoshida K, et al. Development of immunocapture reversetranscription loop-mediated isothermal amplication for the detection of tomatospotted wilt virus from chrysanthemum[J].J Virol Methods,2004,121(1):49-55.
[101] Wang LC, Pan CH, Severinghaus LL, et al. Simultaneous detection anddifferentiation of Newcastle disease and avian influenza viruses usingoligonucleotide microarrays[J].Vet Microbiol,2008,127(3-4):217-226.
[102] Li J, Chen S, Evans DH. Typing and subtyping Influenza virus using DNAmicroarrays and multiplex reverse transcriptase PCR[J]. J Clin Microbiol,2001,39(2):696-704.
[103] Kodihalli S, Sivanandan V, Nagaraja KV, et al. A type-specific avian influenzavirus subunit vaccine for turkeys: induction of Protective immunity to challengeinfection[J]. Vaccine,1994,12(15):1467-1472.
[104]乔传玲,姜永萍,李呈军,等.禽流感重组禽痘病毒rFPV-HA-NA活裁体疫苗的研究[J].免疫学杂志2003,19(1):46-49.
[105]付朝阳,邢大昌,唐秀英,等.高致病力禽流感的流行与防制研究进展[J].中国预防兽医学,2001,23(5):393-396.
[106] Neirynck S,Deroo T,Saelens X, et al. A universal influenza A vaccine based onthe extracellular domain of the M2protein[J]. Nat Med,1999,5(10):1157-1163.
[107] Birzele F, Schaub J, Rust W, et al. Into the unknown: expression profilingwithout genome sequence information in CHO by next generation sequencing [J].Nucleic Acids Res,2010,38(12):3999-4010.
[108] Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAsby stem-loop RT-PCR [J]. Nucleic Acids Res,2005,33(20):e179.