传染性造血器官坏死病毒表面糖蛋白基因核酸疫苗的构建及对虹鳟血液生化指标的影响
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摘要
为研究传染性造血器官坏死病毒(infectious hematopoietic necrosis virus, IHNV)表面糖蛋白(glycoprotein, G)基因核酸疫苗对虹鳟免疫保护效果及血液生化指标的影响,将IHNV G基因克隆至pMD19-T载体中,连接产物在DH5α中进行转化,获取重组质粒pMD19-TG后回收G基因片段。将鉴定正确的G基因片段利用Bam H I和Xho I酶切位点克隆在真核表达载体pVAX1上,构建核酸疫苗pVAX1-G。重组质粒pVAX1-G按照8μg/尾的剂量注射虹鳟设为pVAX1-G组,同时设8μg/尾空质粒组、PBS对照组和空白组,于免疫后21 d,以100倍半数组织培养感染剂量(tissue culture infective dose, TCID50)通过腹腔注射的方式进行攻毒实验,计算核酸疫苗相对保护率(relative percent survival, RPS),攻毒后收集免疫虹鳟血清进行血液指标检测。结果显示,攻毒后虹鳟血清中16项指标中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、总胆红素(TBIL)、总胆汁酸(TBA)、葡萄糖(GLU)、尿素(Urea)、肌酐(CREA)、总蛋白(TP)、白蛋白(ALB)和球蛋白(GLO)与正常虹鳟相比有显著变化,空载组11项指标较pVAX1-G组变化显著;攻毒后14 d pVAX1-G组累积死亡率为19%(19/100),而空载组和PBS对照组分别为62%(62/100)和85%(85/100)。pVAX1-G核酸疫苗对虹鳟免疫保护率为78%。病理学观察发现,免疫pVAX1-G组虹鳟的肝脏、脾脏、肾脏组织未见明显损伤。综上表明,pVAX1-G作为核酸疫苗有助于减轻IHNV对虹鳟的损伤,对IHNV有较好的免疫保护效果。
The aim of this study was to study the effect of infectious hematopoietic necrosis virus(IHNV) nucleic acid vaccine on the immune protection of Oncorhynchus mykiss and serum biochemical indices of Oncorhynchus mykiss. Cloning the glycoprotein gene(G) into pMD19-T vector, we transformed the linked product in DH5α,obtaining recombinant plasmid pMD19-T-G, and recovering the G gene fragment. The identified correct G gene fragment was cloned on eukaryotic expression vector pVAX1 using Bam H I and Xho I cleavage sites to construct nucleic acid vaccine pVAX1-G. The recombinant plasmid pVAX1-G was injected into O. mykiss at a dose of 8μg/ind. as pVAX1-G group, while 8 μg/ind. empty group, PBS control group and blank group were set up.Twenty-one days after immunization, an anti-virus experiment was carried out. After anti-virus, serum was collected and blood indexes were tested. The results showed that there were significant changes in 16 indexes in O.mykiss serum after the attack: glutamic-pyruvic transaminase(ALT), glutamate transaminase(AST), alkaline phosphatase(ALP), total bilirubin(TBIL), total bile acid(TBA), glucose(GLU), the urea(Urea), creatinine(CREA), total protein(TP), albumin(ALB) and globulin(GLO) were compared with normal O. mykiss, and the changes of 11 indexes in no-load group were more significant than those in pVAX1-G group. The cumulative death rate of pVAX1-G group was 19%(19/100)14 days after the attack, while that of no-load group and PBS control group was 62%(62/100) and 85%(85/100), respectively. The protective rate of pVAX1-G nucleic acid vaccine for O. mykiss was 78%. The results showed that pVAX1-G as a nucleic acid vaccine was helpful to reduce IHNV's injury to O. mykiss and had better immune protection effect against IHNV.
The aim of this study was to study the effect of infectious hematopoietic necrosis virus(IHNV) nucleic acid vaccine on the immune protection of Oncorhynchus mykiss and serum biochemical indices of Oncorhynchus mykiss. Cloning the glycoprotein gene(G) into pMD19-T vector, we transformed the linked product in DH5α,obtaining recombinant plasmid pMD19-T-G, and recovering the G gene fragment. The identified correct G gene fragment was cloned on eukaryotic expression vector pVAX1 using Bam H I and Xho I cleavage sites to construct nucleic acid vaccine pVAX1-G. The recombinant plasmid pVAX1-G was injected into O. mykiss at a dose of 8μg/ind. as pVAX1-G group, while 8 μg/ind. empty group, PBS control group and blank group were set up.Twenty-one days after immunization, an anti-virus experiment was carried out. After anti-virus, serum was collected and blood indexes were tested. The results showed that there were significant changes in 16 indexes in O.mykiss serum after the attack: glutamic-pyruvic transaminase(ALT), glutamate transaminase(AST), alkaline phosphatase(ALP), total bilirubin(TBIL), total bile acid(TBA), glucose(GLU), the urea(Urea), creatinine(CREA), total protein(TP), albumin(ALB) and globulin(GLO) were compared with normal O. mykiss, and the changes of 11 indexes in no-load group were more significant than those in pVAX1-G group. The cumulative death rate of pVAX1-G group was 19%(19/100)14 days after the attack, while that of no-load group and PBS control group was 62%(62/100) and 85%(85/100), respectively. The protective rate of pVAX1-G nucleic acid vaccine for O. mykiss was 78%. The results showed that pVAX1-G as a nucleic acid vaccine was helpful to reduce IHNV's injury to O. mykiss and had better immune protection effect against IHNV.
引文
[1]余泽辉.四川地区1株虹鳟(Oncorhynchus mykiss)源传染性造血器官坏死病毒的分离鉴定[D].雅安:四川农业大学,2015.Yu Z H.Isolation and ldentification of an IHNV isolate from rainbow trout(Oncorhynchus mykiss)[D].Yaan:Sichuan Agricultural University,2015(in Chinese).
[2]吴金炉,曾志南.鱼类病毒病与鱼类病毒疫苗[J].海洋科学,1999(4):37-42.Wu J L,Zeng Z N.Fish viral diseases and viral vaccines[J].Marine Sciences,1999(4):37-42(in Chinese).
[3]Kurath G,Leong J C.Characterization of infectious hematopoietic necrosis virus mRNA species reveals a nonvirion rhabdovirus protein[J].Journal of Virology,1985,53(2):462-468.
[4]Corbeil S,Lapatra S E,Anderson E D,et al.Evaluation of the protective immunogenicity of the N,P,M,NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout Oncorhynchus mykiss using DNA vaccines[J].Diseases of Aquatic Organisms,1999,39(1):29-36.
[5]郑风荣.淋巴囊肿病毒(Lymphocystis disease virus)核酸疫苗的免疫效果评价及安全性研究[D].青岛:中国海洋大学,2006.Zheng F R.The research about protective efficiency and safety of DNA vaccine against Lymphocystis disease virus(LCDV)[D].Qingdao:Ocean University of China,2006(in Chinese).
[6]欧阳征亮.鲤春病毒血症病毒(SVCV)糖蛋白DNA疫苗的构建及免疫效果的评估[D].广州:中山大学,2007.Ouyang Z L.Construction of a DNA vaccine encoding spring viremia of carp virus’s glycoprotein and assessment of immune protection in zebrafish(Danio rerio)[D].Guangzhou:Sun Yat-sen University,2007(in Chinese).
[7]乔迁.大菱鲆红体病虹彩病毒核酸疫苗及两种海水名贵鱼类疾病的研究[D].青岛:中国海洋大学,2010.Qiao Q.The study for TRBIV genetic vaccine and the diseases of two rare marine fish species[D].Qingdao:Ocean University of China,2010(in Chinese).
[8]赵巧娥.饲料脂肪水平对鳡幼鱼生长、血液生化指标及消化生理的影响[D].华中农业大学,2011.Zhao Q E.Effect of dietary lipid levels on growth performance,blood biochemical indices and digestive physiology of juvenile yellowcheek carp(Elopichthys bambusa)[D].Huazhong Agricultural University,2011
[9]许品诚,曹萃禾.湖泊围养鱼类血液学指标的初步研究[J].水产学报,1989,13(4):347-352.Xu P C,Cao C H.On hematology of the blood of fishes cultured in the lake pen[J].Journal of Fisheries of China,1989,13(4):347-352(in Chinese).
[10]陈洁,熊邦喜,顾钱洪,等.内源及外源因子对鱼类血液生理生化指标的影响[J].湖北农业科学,2011,50(9):1861-1865.Chen J,Xiong B X,Gu Q H,et al.The effect of endogenous and environment factors on hematology physiological and biochemical indices of fish[J].Hubei Agricultural Sciences,2011,50(9):1861-1865(in Chinese).
[11]Williams K,Blake S,Sweeney A,et al.Multiplex reverse transcriptase PCR assay for simultaneous detection of three fish viruses[J].Journal of Clinical Microbiology,1999,37(12):4139-4141.
[12]Lin L,Zeng X L,Zhang J.Effect of profenofos poisoning on liver lipid peroxidation and liver function in rabbits[J].Chinese Journal of Clinical Rehabilitation,2004,8(21):4380-4381.
[13]张盛华,秦任甲.临床血液流变学指标检测报告单设计不合理应该修正[J].中国医学物理学杂志,2012(1):3181-3183.Zhang S H,Qin R J.The unreasonable setting up for test report of clinical hemorheology indicators should be revised[J].Chinese Journal of Medical Physics,2012(1):3181-3183.
[14]范腾飞,叶应旺,胡王,等.灭活疫苗对异育银鲫败血症血液和生化指标的影响研究[J].淡水渔业,2013,43(4):64-68.Fan T F,Ye Y W,Hu W,et al.Studies on influences of formalin-killed Aeromonas hydrophila on hematological and biochemical parameters in bacterial septicemia Carassius auratus gibelio[J].Freshwater Fisheries,2013,43(4):64-68(in Chinese).
[15]孙金辉,王庆奎,陈成勋,等.嗜水气单胞菌灭活疫苗对虹鳟免疫力和抗病力的影响[J].淡水渔业,2013,43(1):44-49.Sun J H,Wang Q K,Chen C X,et al.Effects of inactivated Aeromonas hydrophila vaccine on immunity and disease resistance of Oncorhynchus mykiss[J].Freshwater Fisheries,2013,43(1):44-49(in Chinese).
[16]郭松林,关瑞章,冯建军,等.嗜水气单胞菌感染对美洲鳗鲡血液和生化指标的影响[J].集美大学学报(自然科学版),2010,15(2):93-97.Guo S L,Guan R Z,Feng J J,et al.Influences of blood and biochemical parameters on American eels(Anguilla rostrata)challenged by Aeromonas hydrophila[J].Journal of Jimei University(Natural Science),2010,15(2):93-97(in Chinese).
[17]Bayne C J,Gerwick L.The acute phase response and innate immunity of fish[J].Developmental&Comparative Immunology,2001,25(8-9):725-743.
[18]徐黎明,赵景壮,卢彤岩,等.一种中国虹鳟传染性造血器官坏死病核酸疫苗及其应用:中国,105816871A[P].2016-08-03.Xu L M,Zhao J Z,Lu T Y,et al.Infectious hematopoietic necrosis(IHN)nucleic acid vaccines for Chinese rainbow trout and application thereof:China,105816871-A[P].2016-08-03(in Chinese).
[19]Fan W,Duan Y J,Huang X L,et al.Pathological changes and etiological diagnosis in farmed adult rainbow trout(Oncorhynchus mykiss)associated with a low mortality[J].Acta Hydrobiologica Sinica,2016,40(2):287-293.
[2]吴金炉,曾志南.鱼类病毒病与鱼类病毒疫苗[J].海洋科学,1999(4):37-42.Wu J L,Zeng Z N.Fish viral diseases and viral vaccines[J].Marine Sciences,1999(4):37-42(in Chinese).
[3]Kurath G,Leong J C.Characterization of infectious hematopoietic necrosis virus mRNA species reveals a nonvirion rhabdovirus protein[J].Journal of Virology,1985,53(2):462-468.
[4]Corbeil S,Lapatra S E,Anderson E D,et al.Evaluation of the protective immunogenicity of the N,P,M,NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout Oncorhynchus mykiss using DNA vaccines[J].Diseases of Aquatic Organisms,1999,39(1):29-36.
[5]郑风荣.淋巴囊肿病毒(Lymphocystis disease virus)核酸疫苗的免疫效果评价及安全性研究[D].青岛:中国海洋大学,2006.Zheng F R.The research about protective efficiency and safety of DNA vaccine against Lymphocystis disease virus(LCDV)[D].Qingdao:Ocean University of China,2006(in Chinese).
[6]欧阳征亮.鲤春病毒血症病毒(SVCV)糖蛋白DNA疫苗的构建及免疫效果的评估[D].广州:中山大学,2007.Ouyang Z L.Construction of a DNA vaccine encoding spring viremia of carp virus’s glycoprotein and assessment of immune protection in zebrafish(Danio rerio)[D].Guangzhou:Sun Yat-sen University,2007(in Chinese).
[7]乔迁.大菱鲆红体病虹彩病毒核酸疫苗及两种海水名贵鱼类疾病的研究[D].青岛:中国海洋大学,2010.Qiao Q.The study for TRBIV genetic vaccine and the diseases of two rare marine fish species[D].Qingdao:Ocean University of China,2010(in Chinese).
[8]赵巧娥.饲料脂肪水平对鳡幼鱼生长、血液生化指标及消化生理的影响[D].华中农业大学,2011.Zhao Q E.Effect of dietary lipid levels on growth performance,blood biochemical indices and digestive physiology of juvenile yellowcheek carp(Elopichthys bambusa)[D].Huazhong Agricultural University,2011
[9]许品诚,曹萃禾.湖泊围养鱼类血液学指标的初步研究[J].水产学报,1989,13(4):347-352.Xu P C,Cao C H.On hematology of the blood of fishes cultured in the lake pen[J].Journal of Fisheries of China,1989,13(4):347-352(in Chinese).
[10]陈洁,熊邦喜,顾钱洪,等.内源及外源因子对鱼类血液生理生化指标的影响[J].湖北农业科学,2011,50(9):1861-1865.Chen J,Xiong B X,Gu Q H,et al.The effect of endogenous and environment factors on hematology physiological and biochemical indices of fish[J].Hubei Agricultural Sciences,2011,50(9):1861-1865(in Chinese).
[11]Williams K,Blake S,Sweeney A,et al.Multiplex reverse transcriptase PCR assay for simultaneous detection of three fish viruses[J].Journal of Clinical Microbiology,1999,37(12):4139-4141.
[12]Lin L,Zeng X L,Zhang J.Effect of profenofos poisoning on liver lipid peroxidation and liver function in rabbits[J].Chinese Journal of Clinical Rehabilitation,2004,8(21):4380-4381.
[13]张盛华,秦任甲.临床血液流变学指标检测报告单设计不合理应该修正[J].中国医学物理学杂志,2012(1):3181-3183.Zhang S H,Qin R J.The unreasonable setting up for test report of clinical hemorheology indicators should be revised[J].Chinese Journal of Medical Physics,2012(1):3181-3183.
[14]范腾飞,叶应旺,胡王,等.灭活疫苗对异育银鲫败血症血液和生化指标的影响研究[J].淡水渔业,2013,43(4):64-68.Fan T F,Ye Y W,Hu W,et al.Studies on influences of formalin-killed Aeromonas hydrophila on hematological and biochemical parameters in bacterial septicemia Carassius auratus gibelio[J].Freshwater Fisheries,2013,43(4):64-68(in Chinese).
[15]孙金辉,王庆奎,陈成勋,等.嗜水气单胞菌灭活疫苗对虹鳟免疫力和抗病力的影响[J].淡水渔业,2013,43(1):44-49.Sun J H,Wang Q K,Chen C X,et al.Effects of inactivated Aeromonas hydrophila vaccine on immunity and disease resistance of Oncorhynchus mykiss[J].Freshwater Fisheries,2013,43(1):44-49(in Chinese).
[16]郭松林,关瑞章,冯建军,等.嗜水气单胞菌感染对美洲鳗鲡血液和生化指标的影响[J].集美大学学报(自然科学版),2010,15(2):93-97.Guo S L,Guan R Z,Feng J J,et al.Influences of blood and biochemical parameters on American eels(Anguilla rostrata)challenged by Aeromonas hydrophila[J].Journal of Jimei University(Natural Science),2010,15(2):93-97(in Chinese).
[17]Bayne C J,Gerwick L.The acute phase response and innate immunity of fish[J].Developmental&Comparative Immunology,2001,25(8-9):725-743.
[18]徐黎明,赵景壮,卢彤岩,等.一种中国虹鳟传染性造血器官坏死病核酸疫苗及其应用:中国,105816871A[P].2016-08-03.Xu L M,Zhao J Z,Lu T Y,et al.Infectious hematopoietic necrosis(IHN)nucleic acid vaccines for Chinese rainbow trout and application thereof:China,105816871-A[P].2016-08-03(in Chinese).
[19]Fan W,Duan Y J,Huang X L,et al.Pathological changes and etiological diagnosis in farmed adult rainbow trout(Oncorhynchus mykiss)associated with a low mortality[J].Acta Hydrobiologica Sinica,2016,40(2):287-293.