我国丁型肝炎病毒全基因克隆及序列分析
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摘要
为构建我国丁型肝炎病毒(HDV)全基因克隆,分别从3份HDV阳性血清中提取病毒RNA,通过反转录-聚合酶链反应(RT-PCR)分段扩增,获得4个相互重叠的DNA片段,将PCR产物测序,利用DNAStar软件拼接,获得HDV全基因组序列;设计引物,用重叠PCR扩增全长HDV片段并连接至克隆载体,构建全基因克隆。结果成功克隆出3株1 675bp的HDV全长基因组。经与GenBank标准序列比对,3株HDV均为基因Ⅰb型,核酸序列同源性达98%以上,与我国Ⅰ型X77627的同源性均达98%以上,与其他基因Ⅰ型的同源性均高于84%。与基因Ⅱ型和Ⅲ型的同源性分别低于77%和66%。本研究构建了3株具有我国代表性的HDV全长基因cDNA克隆,为进一步开展HDV分子生物学研究提供了基础。
The present paper aims to clone and analyze the sequence of full-length cDNA of hepatitis D virus(HDV)in China.HDV RNA was extracted from three HDV seropositive samples.cDNA was obtained by reverse transcription-polymerase chain reaction(RT-PCR).Four overlapped fragments were amplified by nested PCR,and the products were sequenced.The full-length sequences were joined by DNAStar.The fulllength cDNA of HDV was amplified by overlapping PCR using designed primers and connected to the cloning T vector.Three strains of HDV were successfully amplified and cloned.The three HDV strains were all genotype Ib.The nucleic acid sequence homology of the three HDV strains was over 98%.They had over 98%sequence homology to genotype I Chinese strain X77627,and >84% sequence homology to other genotype I strains.They had no more than 77% and 66% sequence homology to genotype II and genotype III respectively.In conclusion,we cloned full-length cDNA of three strains of HDV,which could be helpful for the further study of HDV molecular biology.
The present paper aims to clone and analyze the sequence of full-length cDNA of hepatitis D virus(HDV)in China.HDV RNA was extracted from three HDV seropositive samples.cDNA was obtained by reverse transcription-polymerase chain reaction(RT-PCR).Four overlapped fragments were amplified by nested PCR,and the products were sequenced.The full-length sequences were joined by DNAStar.The fulllength cDNA of HDV was amplified by overlapping PCR using designed primers and connected to the cloning T vector.Three strains of HDV were successfully amplified and cloned.The three HDV strains were all genotype Ib.The nucleic acid sequence homology of the three HDV strains was over 98%.They had over 98%sequence homology to genotype I Chinese strain X77627,and >84% sequence homology to other genotype I strains.They had no more than 77% and 66% sequence homology to genotype II and genotype III respectively.In conclusion,we cloned full-length cDNA of three strains of HDV,which could be helpful for the further study of HDV molecular biology.
引文
[1]李晓娟,况二胜,杨复华.丁型肝炎病毒的分子生物学研究进展[J].中国病毒学,2003,18(3):298-302.
[2]辜文洁,黄维金,周诚,乔杉,伍波,吴星,姚昕,蓝海云,梁争论.不同HDV诊断试剂对HBsAg阳性样品的检测分析[J].中国药事,2011,25(9):866-868,879.
[3]刘善虑,詹美云,谭文杰.中国河南株丁型肝炎病毒全基因组的cDNA克隆和序列分析[J].病毒学报,1994,10(2):97-107.
[4]Makino S,Chang MF,Shieh CK,Kamahora T,Vannier DM,Govindarajan S,Lai MM.Molecular cloning and sequencing of a human hepatitis delta(δ)virus RNA[J].Nature,1987,329(6137):343-346.
[5]Chao YC,Lee CM,Tang HS,Govindarajan S,Lai MM.Molecular cloning and characterization of an isolate of hepatitis delta virus from Taiwan[J].Hepatology,1991,13(2):345-352.
[6]Bergmann KF,Gerin JL.Antigens of hepatitis delta virus in the liver and serum of humans and animals[J].J Infect Dis,1986,154(4):702-706.
[7]Bonino F,Heermann KH,Rizzetto M,Gerlich WH.Hepatitis delta virus:protein composition of delta antigen and its hepatitis B virus-derived envelope[J].J Virol,1986,58(3):945-950.
[8]Chao M,Hsieh SY,Taylor J.Role of two forms of hepatitis delta virus antigen:evidence for a mechanism of self-limiting genome replication[J].J Virol,1990,64(10):5066-5069.
[9]Chang FL,Chen PJ,Tu SJ,Wang CJ,Chen DS.The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus[J].Proc Natl Acad Sci USA,1991,88(19):8490-8494.
[10]刘红,叶荣.病毒蛋白脂酰化及其功能[J].微生物与感染,2014,9(2):122-130.
[11]Zuccola HJ,Rozzelle JE,Lemon SM,Erickson BW,Hogle JM.Structural basis of the oligomerization of hepatitis delta antigen[J].Structure,1998,6(7):821-830.
[12]Xia YP,Lai MM.Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen[J].J Virol,1992,66(11):6641-6648.
[13]Lee CZ,Lin JH,Chao M,McKnight K,Lai MM.RNAbinding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication[J].J Virol,1993,67(4):2221-2227.
[14]Lee CM,Bih FY,Chao YC,Govindarajan S,Lai MM.Evolution of hepatitis delta virus RNA during chronic infection[J].Virology,1992,188(1):265-273.
[15]Casey JL,Brown TL,Colan EJ,Wignall FS,Gerin JL.A genotype of hepatitis D virus that occurs in northern South America[J].Proc Natl Acad Sci USA,1993,90(19):9016-9020.
[2]辜文洁,黄维金,周诚,乔杉,伍波,吴星,姚昕,蓝海云,梁争论.不同HDV诊断试剂对HBsAg阳性样品的检测分析[J].中国药事,2011,25(9):866-868,879.
[3]刘善虑,詹美云,谭文杰.中国河南株丁型肝炎病毒全基因组的cDNA克隆和序列分析[J].病毒学报,1994,10(2):97-107.
[4]Makino S,Chang MF,Shieh CK,Kamahora T,Vannier DM,Govindarajan S,Lai MM.Molecular cloning and sequencing of a human hepatitis delta(δ)virus RNA[J].Nature,1987,329(6137):343-346.
[5]Chao YC,Lee CM,Tang HS,Govindarajan S,Lai MM.Molecular cloning and characterization of an isolate of hepatitis delta virus from Taiwan[J].Hepatology,1991,13(2):345-352.
[6]Bergmann KF,Gerin JL.Antigens of hepatitis delta virus in the liver and serum of humans and animals[J].J Infect Dis,1986,154(4):702-706.
[7]Bonino F,Heermann KH,Rizzetto M,Gerlich WH.Hepatitis delta virus:protein composition of delta antigen and its hepatitis B virus-derived envelope[J].J Virol,1986,58(3):945-950.
[8]Chao M,Hsieh SY,Taylor J.Role of two forms of hepatitis delta virus antigen:evidence for a mechanism of self-limiting genome replication[J].J Virol,1990,64(10):5066-5069.
[9]Chang FL,Chen PJ,Tu SJ,Wang CJ,Chen DS.The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus[J].Proc Natl Acad Sci USA,1991,88(19):8490-8494.
[10]刘红,叶荣.病毒蛋白脂酰化及其功能[J].微生物与感染,2014,9(2):122-130.
[11]Zuccola HJ,Rozzelle JE,Lemon SM,Erickson BW,Hogle JM.Structural basis of the oligomerization of hepatitis delta antigen[J].Structure,1998,6(7):821-830.
[12]Xia YP,Lai MM.Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen[J].J Virol,1992,66(11):6641-6648.
[13]Lee CZ,Lin JH,Chao M,McKnight K,Lai MM.RNAbinding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication[J].J Virol,1993,67(4):2221-2227.
[14]Lee CM,Bih FY,Chao YC,Govindarajan S,Lai MM.Evolution of hepatitis delta virus RNA during chronic infection[J].Virology,1992,188(1):265-273.
[15]Casey JL,Brown TL,Colan EJ,Wignall FS,Gerin JL.A genotype of hepatitis D virus that occurs in northern South America[J].Proc Natl Acad Sci USA,1993,90(19):9016-9020.