表达鹅细小病毒VP3基因重组禽痘病毒的构建
摘要
鹅细小病毒(Goose Parvovirus,GPV)为小鹅瘟的病原体。小鹅瘟主要发生于1月龄内雏鹅和雏番鸭，是以急性肠炎及肝、肾、心实质脏器炎症为特征的烈性传染病，对养鹅业发展造成很大威胁。自1956年我国学者方定一首次发现并分离到GPV后，国外相继也有分离到该病毒的报道，说明该病毒在世界范围内有较为广泛的流行。
目前，对GPV的防制主要是靠疫苗接种。传统疫苗对GPV的预防和控制起到了一定的积极作用，但同时灭活苗存在生产成本高，注射局部炎症反应较强等弊端，弱毒苗又易发生毒力返强等。GPV主要免疫原性蛋白VP3的高度保守性给新型疫苗及分子诊断试剂的研制提供了思路。本研究采用脂质体转染方法，将含有完整GPV H1分离株VP3基因、报告基因LacZ、禽痘病毒早／晚期启动子LP2EP2、痘苗病毒启动子P7.5、P11和FPV-017复制非必须区的转移载体质粒pSY681VP3LacZ与FPV-017共转染鸡胚成纤维细胞，经6轮蚀斑克隆、筛选、表达，PCR鉴定和Dot-ELISA检测，证明该重组病毒已构建成功，并获得了遗传性状稳定的鹅细小病毒VP3基因的重组禽痘病毒。为新型基因工程疫苗的研制奠定了科学的基础，同时也为我国高致病力鹅细小病毒病的防治提供了物质准备。
Goose Parvovirus (GPV) is the pathogen of Goslings plague. This disease is highly fatal to goslings and muscovy ducklings under 1 month of age with the characteristics of acute enteritis and hepatonephric and myocardium lesions, having made large losses to goose breeding industry. In many countries the virus has been isolated after DingYi .F firstly found and isolated it in china, which suggested that this disease has been prevalent in the world.
So far as, prevention of GPV infection mainly depended on vaccines. Although the traditional vaccines have played active role in prevention and controlling the disease, many deficiencies exist still such as inactivated vaccine being expensive and inducing strongly response at the injected tissue, as well as the recovery of the attenuated vaccine. The high conservative main antigenic protein VP3 of GPV provided the access to producing the new type vaccines and diagnostic agent. In this study, the recombinant fowl-poxvirus was transfected into expressing the VP3 gene of isolated GPV H1 strain into the CEF cells with FPV-017 by liposome, which have the LacZ reporter gene, earlier / latter promoters LP2EP2 of FPV, promoters P7.5 and P7.1 of Vaccinia virus, replication unnecessary region of FPV-017. Following 6 cycles screenings, clonings, purification of blue plaques, detection of PCR and Dot-ELISA, which verified the genetic stable VP3-fowlpox virus recombinant constructed successfully. This study provided the theoretical and practical foundation for development of GPV recombinant fowl-poxvirus genetic engineering vaccine, as well as provided substance preparatory for prevention the high mortality GPV.
目前，对GPV的防制主要是靠疫苗接种。传统疫苗对GPV的预防和控制起到了一定的积极作用，但同时灭活苗存在生产成本高，注射局部炎症反应较强等弊端，弱毒苗又易发生毒力返强等。GPV主要免疫原性蛋白VP3的高度保守性给新型疫苗及分子诊断试剂的研制提供了思路。本研究采用脂质体转染方法，将含有完整GPV H1分离株VP3基因、报告基因LacZ、禽痘病毒早／晚期启动子LP2EP2、痘苗病毒启动子P7.5、P11和FPV-017复制非必须区的转移载体质粒pSY681VP3LacZ与FPV-017共转染鸡胚成纤维细胞，经6轮蚀斑克隆、筛选、表达，PCR鉴定和Dot-ELISA检测，证明该重组病毒已构建成功，并获得了遗传性状稳定的鹅细小病毒VP3基因的重组禽痘病毒。为新型基因工程疫苗的研制奠定了科学的基础，同时也为我国高致病力鹅细小病毒病的防治提供了物质准备。
Goose Parvovirus (GPV) is the pathogen of Goslings plague. This disease is highly fatal to goslings and muscovy ducklings under 1 month of age with the characteristics of acute enteritis and hepatonephric and myocardium lesions, having made large losses to goose breeding industry. In many countries the virus has been isolated after DingYi .F firstly found and isolated it in china, which suggested that this disease has been prevalent in the world.
So far as, prevention of GPV infection mainly depended on vaccines. Although the traditional vaccines have played active role in prevention and controlling the disease, many deficiencies exist still such as inactivated vaccine being expensive and inducing strongly response at the injected tissue, as well as the recovery of the attenuated vaccine. The high conservative main antigenic protein VP3 of GPV provided the access to producing the new type vaccines and diagnostic agent. In this study, the recombinant fowl-poxvirus was transfected into expressing the VP3 gene of isolated GPV H1 strain into the CEF cells with FPV-017 by liposome, which have the LacZ reporter gene, earlier / latter promoters LP2EP2 of FPV, promoters P7.5 and P7.1 of Vaccinia virus, replication unnecessary region of FPV-017. Following 6 cycles screenings, clonings, purification of blue plaques, detection of PCR and Dot-ELISA, which verified the genetic stable VP3-fowlpox virus recombinant constructed successfully. This study provided the theoretical and practical foundation for development of GPV recombinant fowl-poxvirus genetic engineering vaccine, as well as provided substance preparatory for prevention the high mortality GPV.
引文
1 陈伯伦，叶本衡，黎杰虹．鹅瘟鸭胚化GD弱毒疫苗的研究．[J]．畜牧兽医学报，第16卷，第四期1985，11：269～273
2 洪峰，共引贤．鹅源鸭瘟与GPV二联弱毒苗的研究．[J]．畜牧兽医报，1991，17(4)：407～410
3 黄奇昌，王红宁．抗小鹅瘟异源高免血清的研制和应用．[J]．四川农业大学学抿1997，15(1)：99～101
4 李茂祥，李俊宝，郑玉美．小鹅瘟病毒纯化及其理化特性的研究．[J]．病毒学报，1990，6：155～159
5 李新华．抗小鹅瘟病毒中和性单克隆抗体的研制及实验防治效果．[J]．中国畜禽传染病，1998，4：247～249
6 李新华．应用斑点酶联免疫吸附实验快速诊断小鹅瘟病毒的研究．[J]．中国兽医科技，1998，28(1)：21～22
7 李忠明，张延龄，徐德启等当代新疫苗朋北京：高等教育出版社．2001：3～7；33～35：126
8 彭万强，朱治远，黄承锋等．GPV鸭胚化弱毒疫苗对雏鹅的免疫研究冲国兽医技，1992，22(4)：9～11
9 秦受建，周阳生．免疫酶斑点法快速检测小鹅瘟病毒．[J]．中国兽医科技，1990，7：28～29
10 斯佩客特D．L，戈德曼R．D，．莱因万德L．A，细胞实验指南．M．北京：科学出版社．2001；27～28；71～72；781～786
11 田晋红等．四川微生物学会论文集．M．1994，7：37～43
12 田丽红，贾永清，王君伟等．鹅细小病毒重组禽痘病毒转移载体的构建．[J]．中国兽医科技，2002，32．(9)：5～7．
13 徐建生，李俊宝，董国雄．小鹅瘟病毒单抗的防治效果．[J]．中国畜禽传染病，第96期1997，5：17～19
14 许振忠,王萌新．抗小鹅瘟高免卵黄抗体的制备和应用．四川畜禽，1995，10：17～18
15 殷震．刘景华．动物病毒学．．M．北京：科学出版社，1997，1603
16 俞翔．抗小鹅瘟免疫血清和免疫卵黄浆的制备及临床应用效果．[J]．冲国兽医科技，1988，12：25～27
17 周阳生，田慧芳，方定一．小鹅瘟疫苗对初生雏鹅的安全性及免疫力试验．[J]．畜牧兽医学报，第16卷1984，10(9)：2～4
18 周阳生，王永坤，方定一．用小鹅瘟疫苗接种母鹅后对其雏鹅天然被动免疫期的测定．[J]中国兽医杂志，1985，11(10)：51～52
19 朱少璇．抗小鹅瘟病毒单克隆抗体及其在防治上的初步应用．[J]．江苏农学院学报，1989，10(3)：41～44
20 张绍杰，童光志，王柳，等．传染性喉气管炎(IL TV)糖蛋白gB在重组禽痘病毒中的表达．中国预防兽医学报，2000,20(3)：205～208
21 Anthony-Cahill S J,Benfied P A,Fairman R,etal.Molecular Characterization of Helix-
Loop-Helixptide. Scince, 1992,255:979～983
22 Astell C R, Terminal Hairpins ofParvovims Genomes and Their Role in DNA Replication. In: Tijssen P.Handbook of parvovims. CRC Press, 1990,59,79
23 Bardona C J,Reed W M, Witter R L,et al. Protein of Turkeys From Hemorrhagic Enteritis With a Recombinant Fowlpox Virus Expressing the Native Hexon of Hemorrhagic Enteritis Virus. Avian Dis, 1999,43:234～244
24 Carter B J,Trempe J P,and Mendelson E.Adeno-Associated Virus Gene Expression and Regulation. In "Handbook of Parvoviruses" (Tijssen P,Ed), CRC Press,Boca Raton,FL. 1990, Vol 1,pp 227～254
25 Chang L, Shi Y,and Shenk T.Adeno-Associated Virus P5 Promoter Contains an Adenovirus E1A Inducible Element and a Binding Site to the Major Late Transcription Factor.[J]Virol, 1989,63:3479～3488
26 Chen K C,Shull B C,Moses E A,et al. Complete Nucleotide Sequence and Genome Organization of Bovine Parvovirus. J Virol, 1986,60:1085～1097
27 Chodosh L A,Carthew R W, and Sharp P A.Asingle Polypeptide Possesses the Binding and Transcription Activities of the Adenovirus Major Late Transcription Factor. Mol Cell Biol. 1986, 6:4723～4733
28 hristensen J,Cotmore S F,Tattersall P.Parvovirus Initiation Factor PIF:a Novel Human DNA-binding Factor Which Coordinately Recognizes two ACGT Mmmotifs[J] Virol.1997, 71(8):5733～5741
29 otmore, S.F,and P.Tattersall. Parvovirus DNA Replication, In M.Depamphilis(ed),DNA Replication in Eukatyotic Cells.Cold Spring Harbor Laboratory Press,Cold SpringgHarbor,N Y.1996,799～813.
30 Deiss V,Tratschin J D, Weiz M, et al. Cloning of the Human Parvovims B19 Genome and Structural Analysis of its Palin Dromic Termini. Virology, 1990,175:247～254
31 Dumas B ,Jourdan M,Pascaud A,et al.Complete Nucleotide Sequence of the Cloned Infections Genome of Junonia Coenia Densovirus Reveals an Organization Unique Among Parvovims. Virology, 1992,191:202～222
32 Gu Z,Plaza S, Perros M,et al.NF-Y Controls Transcription of Minute Virus of Mice P4 Promoter Through Interaction With an Unusual Binding Site.J Virol, 1995,69:239～246
33 Kisary J,Avalosse B,Miller-Faures A,et al. The Genome Structure of a New Chicken Virus Identifies it as a Parvovims.Joumal of General Virology, 1985,66:2259～2263
34 Kisary J.Cross-Neutralization Tests on the Parvoviruses Isolated From Gosling. Avian Pathol, 1974,3:293～296
35 Kisary J.Interaction in Replication Between Goose Parvovims Strain B and Duck Plague Herpesvirus.Arch Virol. 1979,59(1～2):81～88
36 Koonin E V.A Common set of Conserved Motifs in a Vast Variety of Putative Nucleic Acid-Dependent ATPases Including MCM Proteins Involved in the Initiation of Eukaryotic DNA Replication. Nucleic Acids Res, 1993,21:2541～2547
37 Le Gall-Recule G,Jestin W.Biochemical and Genomic Characterization of Muscovy Duck Parvovirus.Arch Virol, 1994,139:121～131
38 Lebovitz R M,Roeder R G.Parvovirus H-I Expression:mapping of the Abundant Cytoplasmic Transcripts and Identification of Promoter Sites and Overlapping Transcription units.J Virol. 1986,(2):271～80
39 Meehan B M,Todd D,Creelan J L,et al. Characterization of Viral DNAs From Cells With Chicken Anaemia Agent:Sequnce Analysis of the Replication Form and Transfection Capabilities of Cloned Genome Fragments.Arch Virol, 124:301～319
40 Mitchell P J,and Tijian R. Transcriptional Regulation in Mammalian Cells by Sequence Specific DNA Binding protein. Scince, 1989,245:371～378
41 NazerianK, Lee L F, Yanagida N. Protection against Marek' sDisease by a Fowlpox Virus Recombinant Expressing the Glycoprotein B of Marek' s Disease Virus.[J]Virol.1992,66(3): 1409～1413.
42 Schettler C H.Isolation of a Highly Pathogenic Virus Fromm Geese With Hepatitis.[J]AviiianDis. 1971,15(2):323～325
43 Seiberg M,Kessler M,Levine A J,Human RNA Polymerase Ⅱ can Prematurely Terminate Transcription of the Adenovirus Type 21ate Transcription Unit at a Precise site that Resembles a Prokaryotic Termination Signal. Virus. Genes. 1987,1(1):97～116
44 Shyder R O,Im D S,and Muzyczka N.Evidence for Covalent Attachment of the Adeno-Associated Virus(AAV) rep Protein to the Ends of the AAV Genome. 1990,64:6024～6213
45 Takehara K,Nakata K,Takizawa C,et al.Expression of Goose Parvovirus Expression System and Establishment of Fluorescent Antibody test to Diagnose Goose Parvovirus Infection.Arch Virol, 1999,144:1639～1845
46 Takehara K,Nishio T,Hayashi Y, et al. An ooutbreak of Goose Parvovirus Infection in Jaoan[J] Vet Med Sci. 1995,57940:777～779
47 Tremper J P,and Carter B J.Alterhate mRNA Splicing is Required for Synthesis of Adeno-Associated Virus VP1Capsid Protein. J Virol, 1988,62:3356～3363
48 Tripathy D N, Schnitzlein W M. Expressing of Avian Influnza Virus Hemagglutinin by Recombinant Fowlpox Virus. Avian Dis, 1991,35:186～191.
49 Tullis G E,Burger L R,Pintel D J,et al. The Minor Capsid VPlof the Autonomous Parvovirus Minute Virus of Mice is Dispensable for Encapsidation of Progeny Single-Stranded DNA But is Required for Infectivity. J Virol, 1993,67:131～141
50 Woiter S,Richards R,Armentrout R W.Cell Cycle-Dependent Replication of the DNA of Minute Virus of Mice,a Parvovirus. Biochim Biophys Acta. 1980,607(3):420～431
51 Zoltan Z,Erdei J,Nagy J,et al.Charaeteristies of the Genome of Goose Parvovirus. Avian Pathol, 1994,23:359～364
2 洪峰，共引贤．鹅源鸭瘟与GPV二联弱毒苗的研究．[J]．畜牧兽医报，1991，17(4)：407～410
3 黄奇昌，王红宁．抗小鹅瘟异源高免血清的研制和应用．[J]．四川农业大学学抿1997，15(1)：99～101
4 李茂祥，李俊宝，郑玉美．小鹅瘟病毒纯化及其理化特性的研究．[J]．病毒学报，1990，6：155～159
5 李新华．抗小鹅瘟病毒中和性单克隆抗体的研制及实验防治效果．[J]．中国畜禽传染病，1998，4：247～249
6 李新华．应用斑点酶联免疫吸附实验快速诊断小鹅瘟病毒的研究．[J]．中国兽医科技，1998，28(1)：21～22
7 李忠明，张延龄，徐德启等当代新疫苗朋北京：高等教育出版社．2001：3～7；33～35：126
8 彭万强，朱治远，黄承锋等．GPV鸭胚化弱毒疫苗对雏鹅的免疫研究冲国兽医技，1992，22(4)：9～11
9 秦受建，周阳生．免疫酶斑点法快速检测小鹅瘟病毒．[J]．中国兽医科技，1990，7：28～29
10 斯佩客特D．L，戈德曼R．D，．莱因万德L．A，细胞实验指南．M．北京：科学出版社．2001；27～28；71～72；781～786
11 田晋红等．四川微生物学会论文集．M．1994，7：37～43
12 田丽红，贾永清，王君伟等．鹅细小病毒重组禽痘病毒转移载体的构建．[J]．中国兽医科技，2002，32．(9)：5～7．
13 徐建生，李俊宝，董国雄．小鹅瘟病毒单抗的防治效果．[J]．中国畜禽传染病，第96期1997，5：17～19
14 许振忠,王萌新．抗小鹅瘟高免卵黄抗体的制备和应用．四川畜禽，1995，10：17～18
15 殷震．刘景华．动物病毒学．．M．北京：科学出版社，1997，1603
16 俞翔．抗小鹅瘟免疫血清和免疫卵黄浆的制备及临床应用效果．[J]．冲国兽医科技，1988，12：25～27
17 周阳生，田慧芳，方定一．小鹅瘟疫苗对初生雏鹅的安全性及免疫力试验．[J]．畜牧兽医学报，第16卷1984，10(9)：2～4
18 周阳生，王永坤，方定一．用小鹅瘟疫苗接种母鹅后对其雏鹅天然被动免疫期的测定．[J]中国兽医杂志，1985，11(10)：51～52
19 朱少璇．抗小鹅瘟病毒单克隆抗体及其在防治上的初步应用．[J]．江苏农学院学报，1989，10(3)：41～44
20 张绍杰，童光志，王柳，等．传染性喉气管炎(IL TV)糖蛋白gB在重组禽痘病毒中的表达．中国预防兽医学报，2000,20(3)：205～208
21 Anthony-Cahill S J,Benfied P A,Fairman R,etal.Molecular Characterization of Helix-
Loop-Helixptide. Scince, 1992,255:979～983
22 Astell C R, Terminal Hairpins ofParvovims Genomes and Their Role in DNA Replication. In: Tijssen P.Handbook of parvovims. CRC Press, 1990,59,79
23 Bardona C J,Reed W M, Witter R L,et al. Protein of Turkeys From Hemorrhagic Enteritis With a Recombinant Fowlpox Virus Expressing the Native Hexon of Hemorrhagic Enteritis Virus. Avian Dis, 1999,43:234～244
24 Carter B J,Trempe J P,and Mendelson E.Adeno-Associated Virus Gene Expression and Regulation. In "Handbook of Parvoviruses" (Tijssen P,Ed), CRC Press,Boca Raton,FL. 1990, Vol 1,pp 227～254
25 Chang L, Shi Y,and Shenk T.Adeno-Associated Virus P5 Promoter Contains an Adenovirus E1A Inducible Element and a Binding Site to the Major Late Transcription Factor.[J]Virol, 1989,63:3479～3488
26 Chen K C,Shull B C,Moses E A,et al. Complete Nucleotide Sequence and Genome Organization of Bovine Parvovirus. J Virol, 1986,60:1085～1097
27 Chodosh L A,Carthew R W, and Sharp P A.Asingle Polypeptide Possesses the Binding and Transcription Activities of the Adenovirus Major Late Transcription Factor. Mol Cell Biol. 1986, 6:4723～4733
28 hristensen J,Cotmore S F,Tattersall P.Parvovirus Initiation Factor PIF:a Novel Human DNA-binding Factor Which Coordinately Recognizes two ACGT Mmmotifs[J] Virol.1997, 71(8):5733～5741
29 otmore, S.F,and P.Tattersall. Parvovirus DNA Replication, In M.Depamphilis(ed),DNA Replication in Eukatyotic Cells.Cold Spring Harbor Laboratory Press,Cold SpringgHarbor,N Y.1996,799～813.
30 Deiss V,Tratschin J D, Weiz M, et al. Cloning of the Human Parvovims B19 Genome and Structural Analysis of its Palin Dromic Termini. Virology, 1990,175:247～254
31 Dumas B ,Jourdan M,Pascaud A,et al.Complete Nucleotide Sequence of the Cloned Infections Genome of Junonia Coenia Densovirus Reveals an Organization Unique Among Parvovims. Virology, 1992,191:202～222
32 Gu Z,Plaza S, Perros M,et al.NF-Y Controls Transcription of Minute Virus of Mice P4 Promoter Through Interaction With an Unusual Binding Site.J Virol, 1995,69:239～246
33 Kisary J,Avalosse B,Miller-Faures A,et al. The Genome Structure of a New Chicken Virus Identifies it as a Parvovims.Joumal of General Virology, 1985,66:2259～2263
34 Kisary J.Cross-Neutralization Tests on the Parvoviruses Isolated From Gosling. Avian Pathol, 1974,3:293～296
35 Kisary J.Interaction in Replication Between Goose Parvovims Strain B and Duck Plague Herpesvirus.Arch Virol. 1979,59(1～2):81～88
36 Koonin E V.A Common set of Conserved Motifs in a Vast Variety of Putative Nucleic Acid-Dependent ATPases Including MCM Proteins Involved in the Initiation of Eukaryotic DNA Replication. Nucleic Acids Res, 1993,21:2541～2547
37 Le Gall-Recule G,Jestin W.Biochemical and Genomic Characterization of Muscovy Duck Parvovirus.Arch Virol, 1994,139:121～131
38 Lebovitz R M,Roeder R G.Parvovirus H-I Expression:mapping of the Abundant Cytoplasmic Transcripts and Identification of Promoter Sites and Overlapping Transcription units.J Virol. 1986,(2):271～80
39 Meehan B M,Todd D,Creelan J L,et al. Characterization of Viral DNAs From Cells With Chicken Anaemia Agent:Sequnce Analysis of the Replication Form and Transfection Capabilities of Cloned Genome Fragments.Arch Virol, 124:301～319
40 Mitchell P J,and Tijian R. Transcriptional Regulation in Mammalian Cells by Sequence Specific DNA Binding protein. Scince, 1989,245:371～378
41 NazerianK, Lee L F, Yanagida N. Protection against Marek' sDisease by a Fowlpox Virus Recombinant Expressing the Glycoprotein B of Marek' s Disease Virus.[J]Virol.1992,66(3): 1409～1413.
42 Schettler C H.Isolation of a Highly Pathogenic Virus Fromm Geese With Hepatitis.[J]AviiianDis. 1971,15(2):323～325
43 Seiberg M,Kessler M,Levine A J,Human RNA Polymerase Ⅱ can Prematurely Terminate Transcription of the Adenovirus Type 21ate Transcription Unit at a Precise site that Resembles a Prokaryotic Termination Signal. Virus. Genes. 1987,1(1):97～116
44 Shyder R O,Im D S,and Muzyczka N.Evidence for Covalent Attachment of the Adeno-Associated Virus(AAV) rep Protein to the Ends of the AAV Genome. 1990,64:6024～6213
45 Takehara K,Nakata K,Takizawa C,et al.Expression of Goose Parvovirus Expression System and Establishment of Fluorescent Antibody test to Diagnose Goose Parvovirus Infection.Arch Virol, 1999,144:1639～1845
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