共表达NDVF、IBDV VP0基因重组鸡痘病毒生物学特性研究
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摘要
将本室构建和筛选的共表达NDV F、IBDV VP0 基因重组鸡痘病毒(重组鸡痘病毒vFV282)经不同的理化因素处理,接种到鸡胚成纤维细胞(CEF)上,观察处理前后重组鸡痘病毒vFV282 对CEF 的毒力和感染滴度,获得该病毒的部分理化学特性。通过鸡胚绒毛尿囊膜传代、CEF 传代和鸡体传代,检测回收毒的毒力、感染滴度,通过PCR 方法检测F、VP0 基因。本试验通过测定不同免疫途径和方法(一次刺种;刺种首免;首免后28d 二免;肌肉注射)和不同时间采血测抗FPV、NDV、IBDV 的中和抗体效价和ConA 刺激脾淋巴细胞反应的净增值,来分析重组鸡痘病毒vFV282 体液免疫和细胞免疫的消长规律和水平。
以上结果为研究重组鸡痘病毒vFV282 免疫水平与免疫攻毒效力的平行关系,为建立效力检验方法和标准,为研究免疫期等奠定了坚实的基础。
Examination of resistance against different physicochemical agents isthe important content of complete identification of a virus. Physicochemicalagents can influence the living and characteristic of virus, even caninactivate virus.
Various kinds of physicochemical agents, such as heat, radiation, pHand chemical inactivator, can change and destruct viral nucleic acids, whichhinder replication, transcription and translation of virus, consequencely.Some physicochemical agents can also influence or destroy capsids orenvelopes of virus, which result in loss of viral infectivity too. Further more,many physicochemical agents have the ability of change and destruct viralnucleic acids and proteins simultaneously.
In summary, it is very significance to understand the effect of variousphysicochemical agents in inactivation of virus. Because various viruseshave different sensitivity to various physicochemical agents, it is veryimportant to keep these agents far away during the preservation of virus.
Belonging to genetic engineering recombinant virus, recombinantFowlpox virus (rFPV) vaccine vFV282 strain has favourableimmunogenicity, which co-express VP0 gene of Infectious Bursal DiseaseVirus (IBDV) and F gene of Newcastle Disease Virus (NDV), can preventeffectively Fowlpox, Newcastle Disease (ND) and Infectious BursalDisease (IBD) in one needle. So it significance to understand someimportant physicochemical characteristic for large scale production,lyophilization, preservation and sterilization of rFPV vaccine vFV282.
In this study, after treatment by various physicochemical
agents, including temperature, HCl (pH3), NaOH (pH9), ether,chloroform, and trypsogen, rFPV vFV282 was inoculated in CEF.And the changes of virulence and infectivity of vFV282 wereevaluated by examined TCID50, in order to analyze the influence of theseagents on vFV282. The results indicated that the change of pH(3.0~9.0) didn’t lead to apparent change of virulence and infectivity.However, vFV282 could be inactivated by 55℃for 60min or 60℃for 15min. vFV282 was sensitive to chloroform, so that infectivitydropped 5.5 log10TCID50 after treatment with 5% chloroform for 10 min.Treated with 1% trypsogen in 37℃for an hour, TCID50 reduced 2.55log10TCID50. These results indicated the susceptiveness of vFV282towards trypsogen. Results also suggested the resistance of vFV282towards ether, for infectivity reduced only 1.0 log10TCID50 after treatmentwith ether for 24 hours.
In order to evaluate the inherent stability of vFV282, we examined theF gene of NDV and VP0 gene of IBDV carried by vFV282. First, vFV282was cultivated on SPF embryoes of chicken 10 passages continuously. Then,passage 3rd, 6th, 10th were identified by PCR and examination on TCID50of vFV282. Secondly, vFV282 was cultivated on CEF 30 passagescontinuously. And passage 1st, 5th, 10th, 15th, 20th, 25th, 30th wereidentified. Finally, vFV282 was cultivated 8 passages on chickenscontinuously, and 3rd, 5th, 8th were identified. The results indicated thatlost of VP0、F gene did not happen, and TCID50 did not change.Furthermore, VP0 gene and F gene were cloned into pMD18-T vector andsequenced, respectively. The result confirmed the stability of these twogenes.
Previous studies proved rFPV vFV282 could replicated in chickens,expressed and presented F gene of NDV, VP0 gene of IBDV and wholegenes of FPV, and induced the specific antibodies, which could enhancecellular immunity of chicken. In this study, chickens were inoculated usingvFV282 by various vaccination strategies. And neutralizing antibodiesagainst FPV, NDV, IBDV and lymphoproliferation of vaccinated chickenwere examined respectively, in order to analyze the profile and intensity ofspecific humoral and cellular immune responses.
Chickens of 7-days old were divided into 4 groups, such as controlgroup, intramuscular injection group, intradermal injection once group,intradermal injection twice group. Experimental groups inoculated a dozeof vFV282 (≥105.5TCID50), and control group was inoculated saline.Post-vaccinated, blood and spleen of chickens were collected on 7d, 14d,21d, 28d, 35d, 42d, 49d and 56d. Specific neutralizing antibodies anti-FPV,anti-NDV and anti-IBDV were detected respectively. And Tlymphoproliferations were also examined by MTT assay.
The results showed that the specific neutralizing antibodies ofchickens belonging to intradermal injection group and intramuscularinjection group rose rapidly from 7dpv, arrived peak on 14~21dpv, anddropped from 28dpv, but still kept a favourable level on 42dpv. Inintradermal injection group, anti-FPV neutralizing antibody rose veryquickly from 7dpv, arrived top level on 14dpv (1:44.67), dropped from28dpv (1:17.70), still kept calm on 42dpv (1:9.25); anti-NDV neutralizingantibody rose directly from 7dpv, peaked on 14dpv (1:89.13), descendedfrom 28dpv (1:19.63), still had a weak extent (1:14.19); anti-IBDVantibody rocketed from 7dpv, reached top level on 21dpv (1:89.13),
以上结果为研究重组鸡痘病毒vFV282 免疫水平与免疫攻毒效力的平行关系,为建立效力检验方法和标准,为研究免疫期等奠定了坚实的基础。
Examination of resistance against different physicochemical agents isthe important content of complete identification of a virus. Physicochemicalagents can influence the living and characteristic of virus, even caninactivate virus.
Various kinds of physicochemical agents, such as heat, radiation, pHand chemical inactivator, can change and destruct viral nucleic acids, whichhinder replication, transcription and translation of virus, consequencely.Some physicochemical agents can also influence or destroy capsids orenvelopes of virus, which result in loss of viral infectivity too. Further more,many physicochemical agents have the ability of change and destruct viralnucleic acids and proteins simultaneously.
In summary, it is very significance to understand the effect of variousphysicochemical agents in inactivation of virus. Because various viruseshave different sensitivity to various physicochemical agents, it is veryimportant to keep these agents far away during the preservation of virus.
Belonging to genetic engineering recombinant virus, recombinantFowlpox virus (rFPV) vaccine vFV282 strain has favourableimmunogenicity, which co-express VP0 gene of Infectious Bursal DiseaseVirus (IBDV) and F gene of Newcastle Disease Virus (NDV), can preventeffectively Fowlpox, Newcastle Disease (ND) and Infectious BursalDisease (IBD) in one needle. So it significance to understand someimportant physicochemical characteristic for large scale production,lyophilization, preservation and sterilization of rFPV vaccine vFV282.
In this study, after treatment by various physicochemical
agents, including temperature, HCl (pH3), NaOH (pH9), ether,chloroform, and trypsogen, rFPV vFV282 was inoculated in CEF.And the changes of virulence and infectivity of vFV282 wereevaluated by examined TCID50, in order to analyze the influence of theseagents on vFV282. The results indicated that the change of pH(3.0~9.0) didn’t lead to apparent change of virulence and infectivity.However, vFV282 could be inactivated by 55℃for 60min or 60℃for 15min. vFV282 was sensitive to chloroform, so that infectivitydropped 5.5 log10TCID50 after treatment with 5% chloroform for 10 min.Treated with 1% trypsogen in 37℃for an hour, TCID50 reduced 2.55log10TCID50. These results indicated the susceptiveness of vFV282towards trypsogen. Results also suggested the resistance of vFV282towards ether, for infectivity reduced only 1.0 log10TCID50 after treatmentwith ether for 24 hours.
In order to evaluate the inherent stability of vFV282, we examined theF gene of NDV and VP0 gene of IBDV carried by vFV282. First, vFV282was cultivated on SPF embryoes of chicken 10 passages continuously. Then,passage 3rd, 6th, 10th were identified by PCR and examination on TCID50of vFV282. Secondly, vFV282 was cultivated on CEF 30 passagescontinuously. And passage 1st, 5th, 10th, 15th, 20th, 25th, 30th wereidentified. Finally, vFV282 was cultivated 8 passages on chickenscontinuously, and 3rd, 5th, 8th were identified. The results indicated thatlost of VP0、F gene did not happen, and TCID50 did not change.Furthermore, VP0 gene and F gene were cloned into pMD18-T vector andsequenced, respectively. The result confirmed the stability of these twogenes.
Previous studies proved rFPV vFV282 could replicated in chickens,expressed and presented F gene of NDV, VP0 gene of IBDV and wholegenes of FPV, and induced the specific antibodies, which could enhancecellular immunity of chicken. In this study, chickens were inoculated usingvFV282 by various vaccination strategies. And neutralizing antibodiesagainst FPV, NDV, IBDV and lymphoproliferation of vaccinated chickenwere examined respectively, in order to analyze the profile and intensity ofspecific humoral and cellular immune responses.
Chickens of 7-days old were divided into 4 groups, such as controlgroup, intramuscular injection group, intradermal injection once group,intradermal injection twice group. Experimental groups inoculated a dozeof vFV282 (≥105.5TCID50), and control group was inoculated saline.Post-vaccinated, blood and spleen of chickens were collected on 7d, 14d,21d, 28d, 35d, 42d, 49d and 56d. Specific neutralizing antibodies anti-FPV,anti-NDV and anti-IBDV were detected respectively. And Tlymphoproliferations were also examined by MTT assay.
The results showed that the specific neutralizing antibodies ofchickens belonging to intradermal injection group and intramuscularinjection group rose rapidly from 7dpv, arrived peak on 14~21dpv, anddropped from 28dpv, but still kept a favourable level on 42dpv. Inintradermal injection group, anti-FPV neutralizing antibody rose veryquickly from 7dpv, arrived top level on 14dpv (1:44.67), dropped from28dpv (1:17.70), still kept calm on 42dpv (1:9.25); anti-NDV neutralizingantibody rose directly from 7dpv, peaked on 14dpv (1:89.13), descendedfrom 28dpv (1:19.63), still had a weak extent (1:14.19); anti-IBDVantibody rocketed from 7dpv, reached top level on 21dpv (1:89.13),
引文
[1] 李永明,吴彤,周碧君,等.贵州省鸡产蛋下降综合征病的病原分离[J]. 中国兽医杂志,1999,23(4):12～14
[2] 周继勇,李建荣. 传染性法氏囊病病毒变异株弱毒的遗传稳定性与免疫原性. 中国兽医学报,2000,20(4):317～320
[3] 王云峰,智海东,王玫,等表达传染性喉气管炎病毒gB基因重组鸡痘病毒疫苗的遗传稳定性评价. 中国预防兽医学报2001,23(6): 426～429
[4] 丁巧玲,陈溥言. 表达MDV GB 基因的重组鸡痘病毒的免疫效果研究. 南京农业大学学报2002,25(4):77～80
[5] Fenner F,Henderson DA,Arita L,etal.Smallpox and its eradication.Geneva:World Health Organization. 1988.
[6] 殷震,刘景华.动物病毒学.第二版.[M]北京:科学出版社,1997
[7] Boyle D B,Heine H G.Recombinant fowlpox virus vaccines for poultry[J].Immunol Coll Boil.1993,71:391～397.
[8] Levine MM,Woodrow GC,Kaper JB,etal.New generation vaccines.Marcel Dekker,Inc,.Newyork USA. 1997.
[9] Plotkin SA,Orenstein WA.Vaccines.W.B.Saunders Company,Philadephia,USA. 1999.
[10] Ogawa r,etal.Recombinant fowlpox viruses inducing protective against Newcastle disease and fowlpox viruses.vaccine,1990,8:486～490.
[11] Mackett m, Smith G, Moss B. Vaccinia Virus: A Selectable Enkaryotic Clonmy and Expression Vector[J].Proc. Natl Acad, Sci, 1982, 79:7415～7419
[12] Panicali D, Paoletti E. Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus[J]. Proc. Natl. Acad. Sci. U. S. A 1982,79(16): 4927～4931
[13] Mcmillen J, Cochram M, Junker D. The Safe and Effective Use of Fowlpox as a Vector for Poultry Vaccines[J]. Dev. Biol. Standard, 1994, 82: 137～145
[14] Yanagida n,etal.recombinant fowlpox viruses expressing the glycoprotein b homolog and the p38 gene of marek’s disease virus.j virol,1992,66:1409～1413
[15] Taylor j,etal.recombinant fowlpox virus inducing protectable immunity in nonavian species.vaccine,1988,6:497～503.
[16] Cadoz M,Strady A,Meiqner B,etal. Immunization of human using a novel vector:Canarypox expressing rabies glycoprotein.Lancet, 1992.339:1429～1432.
[17] 马海伦,孙朝晖,陆柔剑等。1999,同时表达多种外源基因的非 复制型重组痘苗病毒的构建。病毒学报,15(1):21～28
[18] Taylor j,etal.Nonreplicatng viral vector as potential vaccines:recombinant canarypox virus expressing measles virus fusion(f) and hemagglutinin(ha) glycoproteins.virology.
[19] 彭大新刘秀梵程坚共表达mdv 糖蛋白和鸡γ干扰素基因的重 组鸡痘病毒的构建微生物学报2002,42(5),:611～615
[20] 沈瑞忠,王笑梅,陈忠斌,等。表达传染性法氏囊病病毒VP2 基 因的重组鸡痘病毒的构建及其免疫保护性的研究(J)。中国畜禽 传染病,1998,20(2):65～67
[21] 夏志平,共表达新城疫F 基因、传染性囊病毒VP0 基因的重组鸡 痘病毒构建及其实验免疫研究[D], 博士论文,2002
[22] Fenner F,Henderson DA,Arita L,etal. Smallpox and its eradication.Geneva:World Health Organization. 1988.
[23] 刘毅,金宁一,古长庆,等。IBDV VP2-VP234 基因疫苗表达质 粒的构建与表达(J)。中国兽医学报,2000,20(4):321～323。
[24] Moss B.Poxviridae and their replication (2nd)[M],Rarom Press .1990,11～18.
[25] Hirt p. Hiller g, Wittik r. Localizationg and fine structure of vaccinia virus gene encoding an envelop antigen[J],J virol ,1986,58,757～764
[26] Holzer G W,Grditschenberger W ,Mayrhofer J A.etal.Dominant bost range selection of vaccinia recombinants by rescue of an essential gene [J].Virology,1998,249:160～166
[27] Kumar S,Boyle D B.Activity of a fowlpox virus in late promoter in vaccinia and fowlpox virus recombinants[J].Arch Virol,1990,112:139～148.
[28] Spehner D R,Drillien R,Lecocy J P.Construction of fowlpox virus vectors with intergenic insertions:expression of the beta –galactosidase gene and the measles virus fusion gene[J],Virol.1990,64(2):527～533.
[29] Boursnell M E G,Green P F ,Camphell J I A ,etal.Insertion of fusion gene of Newcastle disease virus into a non-essential region in the terminal repeats of fowlpox virus and demenstration of protective immunity induued by the recombinant[J].J Gen Virol ,1990. 71:621～ 628.
[30] Ogawa R,Calvert L G,Yanagida N .Insertional inactivation of a fowlpox virus homologue of the vaccinia virus F12L gene inhibits the release of enveloped virions[J],J Gen Virol ,1993,74:55～64
[31] Heine H G ,Boyle D B .Infectious bursal disease virus structural protein VP2 expressed by fowlpox virus recombinant confers protection against disease in chickens[J],Arch Virol ,1993,131:277～ 292.
[32] Iritani Y,Aoyama S,Takigami S,etal.Antibody response to Newcastle disease virus of recombinant fowlpox expressing a hemagglutinin-neuraminidase of NDV into chickens in the presence of antibody to NDV and FPV[J],Avian Dis,1991,34:185～192
[33] Yu Q Z,Thomas b, David T K,etal.Protection against turkey rhinotracheitis (trtv)indicated by a fowlpox virus recombinant expressing the trtv fusion glycoprotein[J].Vaccine,1994, 12(6):569～ 573.
[34] Cardona C J,Reed W M,Witter R I,etal.Protection of turkeys from hemorrhagic enteritis with a recombinant fowl poxvirus expressing the native hexon of hemorrhagic enteritis virus[j].Avian Dis,1999,43(2):234～244.
[35] Leong C J,Ramsay A J,Boyle D B,etal.Selection induction of immune response by cytokines coexpressed in recombinant fowlpox virus[J]. J Virol,1994,68:8125～8130
[36] Couper B E H,Teo T,Boyle D B .Restriction endonuclease mapping of fowlpox virus genome[F].Virology,1990,179:159～167
[37] Czyz A,Kolacz E,Angerer,etal. Expression of activation antigens on lymphocyte surface and circulating platelet-leukocyte aggregates in ischaemic heart disease. Kardiol Pol. 2005 Mar;62(3):189～202.
[38] Boyle d b,couper e h.construction of recombinant fowlpox virus as vectors for poultry vaccines.virus reseach.1998,10:343～356.
[39] 王明俊等:兽医生物制品学[M],中国农业出版社,1996。
[40] Taylor j,paoletti e.fowlpox virus as a vector in non-avianspecies.vaccine,1988,6:466～468
[41] Burgos JS,Ramirez C,Tenorio R,etal. Influence of reagents formulation on real-time PCR parameters.Mol Cell Probes. 2002 Aug;16(4):257～ 60.
[42] 夏志平、金宁一、金扩世等。共表达新城疫病毒F 基因、传染性 法氏囊病毒VP0 基因的重组鸡痘病毒的抗原性和免疫原性[J]。中 国兽医学报,2003,23(3):214～217
[43] Nakajima K. The mechanism of antigenic shift and drift of human influenza virus. Nippon Rinsho. 2003 Nov;61(11):1897～903.
[44] 王云峰,智海东,王玫等表达传染性喉气管炎病毒gB 基因重组 鸡痘病毒疫苗的遗传稳定性评价中国预防兽医学报2001,23 (6):426～429。
[45] 李萍萍,陈乃民,应保龄等重组痘苗狂犬病毒糖蛋白的构建、表达 及其遗传稳定性研究微生物学杂志2002 22(2): 18～20
[46] [ 美] J . 萨姆布鲁克等. 分子克隆实验指南[ M ]. 科学出版 社,1992,55～56,318～324.
[47] Taylor J etal.Efficacy studies on acanarypox rabies recombinant virus[J] .Vaccine,1991,9(3):190～193.
[48] 马文军,于康震.禽流感疫苗[J].中国预防兽医报,1999,21(5):380～400.
[49] Wolff JA,Malone R,Williams P,etal.Direct gene transfer into mouse muscle in vivo[J].Science ,1990,247:1465～1468.
[50] Kenessey A,Ojamaa K. Ligand-mediated decrease of thyroid hormone receptor-alpha1 in cardiomyocytes by proteosome-dependent
[2] 周继勇,李建荣. 传染性法氏囊病病毒变异株弱毒的遗传稳定性与免疫原性. 中国兽医学报,2000,20(4):317～320
[3] 王云峰,智海东,王玫,等表达传染性喉气管炎病毒gB基因重组鸡痘病毒疫苗的遗传稳定性评价. 中国预防兽医学报2001,23(6): 426～429
[4] 丁巧玲,陈溥言. 表达MDV GB 基因的重组鸡痘病毒的免疫效果研究. 南京农业大学学报2002,25(4):77～80
[5] Fenner F,Henderson DA,Arita L,etal.Smallpox and its eradication.Geneva:World Health Organization. 1988.
[6] 殷震,刘景华.动物病毒学.第二版.[M]北京:科学出版社,1997
[7] Boyle D B,Heine H G.Recombinant fowlpox virus vaccines for poultry[J].Immunol Coll Boil.1993,71:391～397.
[8] Levine MM,Woodrow GC,Kaper JB,etal.New generation vaccines.Marcel Dekker,Inc,.Newyork USA. 1997.
[9] Plotkin SA,Orenstein WA.Vaccines.W.B.Saunders Company,Philadephia,USA. 1999.
[10] Ogawa r,etal.Recombinant fowlpox viruses inducing protective against Newcastle disease and fowlpox viruses.vaccine,1990,8:486～490.
[11] Mackett m, Smith G, Moss B. Vaccinia Virus: A Selectable Enkaryotic Clonmy and Expression Vector[J].Proc. Natl Acad, Sci, 1982, 79:7415～7419
[12] Panicali D, Paoletti E. Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus[J]. Proc. Natl. Acad. Sci. U. S. A 1982,79(16): 4927～4931
[13] Mcmillen J, Cochram M, Junker D. The Safe and Effective Use of Fowlpox as a Vector for Poultry Vaccines[J]. Dev. Biol. Standard, 1994, 82: 137～145
[14] Yanagida n,etal.recombinant fowlpox viruses expressing the glycoprotein b homolog and the p38 gene of marek’s disease virus.j virol,1992,66:1409～1413
[15] Taylor j,etal.recombinant fowlpox virus inducing protectable immunity in nonavian species.vaccine,1988,6:497～503.
[16] Cadoz M,Strady A,Meiqner B,etal. Immunization of human using a novel vector:Canarypox expressing rabies glycoprotein.Lancet, 1992.339:1429～1432.
[17] 马海伦,孙朝晖,陆柔剑等。1999,同时表达多种外源基因的非 复制型重组痘苗病毒的构建。病毒学报,15(1):21～28
[18] Taylor j,etal.Nonreplicatng viral vector as potential vaccines:recombinant canarypox virus expressing measles virus fusion(f) and hemagglutinin(ha) glycoproteins.virology.
[19] 彭大新刘秀梵程坚共表达mdv 糖蛋白和鸡γ干扰素基因的重 组鸡痘病毒的构建微生物学报2002,42(5),:611～615
[20] 沈瑞忠,王笑梅,陈忠斌,等。表达传染性法氏囊病病毒VP2 基 因的重组鸡痘病毒的构建及其免疫保护性的研究(J)。中国畜禽 传染病,1998,20(2):65～67
[21] 夏志平,共表达新城疫F 基因、传染性囊病毒VP0 基因的重组鸡 痘病毒构建及其实验免疫研究[D], 博士论文,2002
[22] Fenner F,Henderson DA,Arita L,etal. Smallpox and its eradication.Geneva:World Health Organization. 1988.
[23] 刘毅,金宁一,古长庆,等。IBDV VP2-VP234 基因疫苗表达质 粒的构建与表达(J)。中国兽医学报,2000,20(4):321～323。
[24] Moss B.Poxviridae and their replication (2nd)[M],Rarom Press .1990,11～18.
[25] Hirt p. Hiller g, Wittik r. Localizationg and fine structure of vaccinia virus gene encoding an envelop antigen[J],J virol ,1986,58,757～764
[26] Holzer G W,Grditschenberger W ,Mayrhofer J A.etal.Dominant bost range selection of vaccinia recombinants by rescue of an essential gene [J].Virology,1998,249:160～166
[27] Kumar S,Boyle D B.Activity of a fowlpox virus in late promoter in vaccinia and fowlpox virus recombinants[J].Arch Virol,1990,112:139～148.
[28] Spehner D R,Drillien R,Lecocy J P.Construction of fowlpox virus vectors with intergenic insertions:expression of the beta –galactosidase gene and the measles virus fusion gene[J],Virol.1990,64(2):527～533.
[29] Boursnell M E G,Green P F ,Camphell J I A ,etal.Insertion of fusion gene of Newcastle disease virus into a non-essential region in the terminal repeats of fowlpox virus and demenstration of protective immunity induued by the recombinant[J].J Gen Virol ,1990. 71:621～ 628.
[30] Ogawa R,Calvert L G,Yanagida N .Insertional inactivation of a fowlpox virus homologue of the vaccinia virus F12L gene inhibits the release of enveloped virions[J],J Gen Virol ,1993,74:55～64
[31] Heine H G ,Boyle D B .Infectious bursal disease virus structural protein VP2 expressed by fowlpox virus recombinant confers protection against disease in chickens[J],Arch Virol ,1993,131:277～ 292.
[32] Iritani Y,Aoyama S,Takigami S,etal.Antibody response to Newcastle disease virus of recombinant fowlpox expressing a hemagglutinin-neuraminidase of NDV into chickens in the presence of antibody to NDV and FPV[J],Avian Dis,1991,34:185～192
[33] Yu Q Z,Thomas b, David T K,etal.Protection against turkey rhinotracheitis (trtv)indicated by a fowlpox virus recombinant expressing the trtv fusion glycoprotein[J].Vaccine,1994, 12(6):569～ 573.
[34] Cardona C J,Reed W M,Witter R I,etal.Protection of turkeys from hemorrhagic enteritis with a recombinant fowl poxvirus expressing the native hexon of hemorrhagic enteritis virus[j].Avian Dis,1999,43(2):234～244.
[35] Leong C J,Ramsay A J,Boyle D B,etal.Selection induction of immune response by cytokines coexpressed in recombinant fowlpox virus[J]. J Virol,1994,68:8125～8130
[36] Couper B E H,Teo T,Boyle D B .Restriction endonuclease mapping of fowlpox virus genome[F].Virology,1990,179:159～167
[37] Czyz A,Kolacz E,Angerer,etal. Expression of activation antigens on lymphocyte surface and circulating platelet-leukocyte aggregates in ischaemic heart disease. Kardiol Pol. 2005 Mar;62(3):189～202.
[38] Boyle d b,couper e h.construction of recombinant fowlpox virus as vectors for poultry vaccines.virus reseach.1998,10:343～356.
[39] 王明俊等:兽医生物制品学[M],中国农业出版社,1996。
[40] Taylor j,paoletti e.fowlpox virus as a vector in non-avianspecies.vaccine,1988,6:466～468
[41] Burgos JS,Ramirez C,Tenorio R,etal. Influence of reagents formulation on real-time PCR parameters.Mol Cell Probes. 2002 Aug;16(4):257～ 60.
[42] 夏志平、金宁一、金扩世等。共表达新城疫病毒F 基因、传染性 法氏囊病毒VP0 基因的重组鸡痘病毒的抗原性和免疫原性[J]。中 国兽医学报,2003,23(3):214～217
[43] Nakajima K. The mechanism of antigenic shift and drift of human influenza virus. Nippon Rinsho. 2003 Nov;61(11):1897～903.
[44] 王云峰,智海东,王玫等表达传染性喉气管炎病毒gB 基因重组 鸡痘病毒疫苗的遗传稳定性评价中国预防兽医学报2001,23 (6):426～429。
[45] 李萍萍,陈乃民,应保龄等重组痘苗狂犬病毒糖蛋白的构建、表达 及其遗传稳定性研究微生物学杂志2002 22(2): 18～20
[46] [ 美] J . 萨姆布鲁克等. 分子克隆实验指南[ M ]. 科学出版 社,1992,55～56,318～324.
[47] Taylor J etal.Efficacy studies on acanarypox rabies recombinant virus[J] .Vaccine,1991,9(3):190～193.
[48] 马文军,于康震.禽流感疫苗[J].中国预防兽医报,1999,21(5):380～400.
[49] Wolff JA,Malone R,Williams P,etal.Direct gene transfer into mouse muscle in vivo[J].Science ,1990,247:1465～1468.
[50] Kenessey A,Ojamaa K. Ligand-mediated decrease of thyroid hormone receptor-alpha1 in cardiomyocytes by proteosome-dependent