口蹄疫疫苗非抗原蛋白对146S抗原免疫效果的影响
|
摘要
为探究非抗原蛋白对口蹄疫病毒A型(FMDV-A) 146S抗原免疫效果的影响,本研究以小鼠和猪为试验动物,分别制备5组小鼠注射用样品:A组(3μg 146S抗原)、B组(3μg 146S抗原+25μg非抗原蛋白)、C组(3μg 146S抗原+50μg非抗原蛋白)、D组(3μg 146S抗原+100μg非抗原蛋白)及E组(空白对照,PBS);同时制备4组猪注射用样品:A组(18μg 146S抗原)、B组(18μg 146S抗原+400μg非抗原蛋白)、C(18μg 146S抗原+4 000μg非抗原蛋白)及D组(空白对照,PBS)。免疫接种试验动物后,通过淋巴细胞增殖试验及实时荧光定量PCR评价比较不同疫苗样品组小鼠产生的细胞免疫应答水平,同时通过液相阻断ELISA方法检测、评价各试验组动物(小鼠和猪)产生的体液免疫应答水平。小鼠试验结果显示,在免疫后3个检测时间点内,4个试验组中C组的平均抗体水平最高,平均抗体效价分别为4.13、5.83和5.50,而D组的抗体水平最低,平均抗体效价分别为3.46、5.16和4.46;C组细胞增殖能力及Th-1型细胞因子IL-6、IFN-β和TNF-αmRNA表达量均高于其他组。猪试验结果显示,A组和B组间平均抗体水平差异不显著(P>0.05),但A组和B组的平均抗体效价均极显著高于C组(P<0.01)。综合上述结果表明,一定范围内少量非抗原蛋白对FMDV-A 146S抗原的免疫效果没有影响,而高浓度的非抗原蛋白则抑制FMDV-A 146S抗原的免疫效果。
In order to explore the effect of non-antigen protein on the immune effect of foot-and-mouth disease virus type A(FMDV-A) 146 S antigen,mice and pigs were used as experimental animals.5 groups of mice for injection were prepared:Group A(3 μg 146 S antigen),group B(3 μg 146 S antigen+25 μg non-antigenic protein),group C(3 μg 146 S antigen+50 μg non-antigenic protein),group D(3 μg 146 S antigen+100 μg non-antigenic protein) and group E(blank control,PBS).At the same time,four groups of pig injection samples were prepared:Group A(18 μg 146 S antigen),group B(18 μg 146 S antigen+400 μg non-antigenic protein),group C(18 μg 146 S antigen+4 000 μg non-antigenic protein) and group D(blank control,PBS).The cell immune response levels of mice in different vaccine sample groups were compared by lymphocyte proliferation assay and Real-time PCR.The level of humoral immune response produced by the animals(mice and pigs) of each test group was detected and evaluated by a liquid phase blocking ELISA method.The results of mice test showed that the average antibody level of group C was the highest in four test groups at the three test time points after immunization,and the average antibody titers were 4.13,5.83 and 5.50,respectively,while the antibody of group D was the lowest,the average antibody titer average antibody titers were 3.46,5.16 and 4.46 in the antibody detection,respectively;The cell proliferation ability and Th-1 type cytokine IL-6,IFN-β and TNF-α mRNA expression in group C were higher than other groups.The results of pigs test showed that the average antibody level between groups A and B was not significantly different(P>0.05),but the average antibody titers of groups A and B were extremely significantly higher than that of group C(P<0.01).The above results indicated that a small amount of non-antigenic protein had no effect on the immune effect of FMDV-A 146 S antigen within a certain range,while a high concentration of non-antigenic protein inhibited the immune effect of FMDV-A 146 S antigen.
In order to explore the effect of non-antigen protein on the immune effect of foot-and-mouth disease virus type A(FMDV-A) 146 S antigen,mice and pigs were used as experimental animals.5 groups of mice for injection were prepared:Group A(3 μg 146 S antigen),group B(3 μg 146 S antigen+25 μg non-antigenic protein),group C(3 μg 146 S antigen+50 μg non-antigenic protein),group D(3 μg 146 S antigen+100 μg non-antigenic protein) and group E(blank control,PBS).At the same time,four groups of pig injection samples were prepared:Group A(18 μg 146 S antigen),group B(18 μg 146 S antigen+400 μg non-antigenic protein),group C(18 μg 146 S antigen+4 000 μg non-antigenic protein) and group D(blank control,PBS).The cell immune response levels of mice in different vaccine sample groups were compared by lymphocyte proliferation assay and Real-time PCR.The level of humoral immune response produced by the animals(mice and pigs) of each test group was detected and evaluated by a liquid phase blocking ELISA method.The results of mice test showed that the average antibody level of group C was the highest in four test groups at the three test time points after immunization,and the average antibody titers were 4.13,5.83 and 5.50,respectively,while the antibody of group D was the lowest,the average antibody titer average antibody titers were 3.46,5.16 and 4.46 in the antibody detection,respectively;The cell proliferation ability and Th-1 type cytokine IL-6,IFN-β and TNF-α mRNA expression in group C were higher than other groups.The results of pigs test showed that the average antibody level between groups A and B was not significantly different(P>0.05),but the average antibody titers of groups A and B were extremely significantly higher than that of group C(P<0.01).The above results indicated that a small amount of non-antigenic protein had no effect on the immune effect of FMDV-A 146 S antigen within a certain range,while a high concentration of non-antigenic protein inhibited the immune effect of FMDV-A 146 S antigen.
引文
[1] 陈溥言.兽医传染病学[M].北京:中国农业出版社,2006. CHEN F Y.Veterinary Infectious Diseases[M].Beijing:China Agriculture Press,2006.(in Chinese)
[2] KNIGHT-JONES T J D,RUSHTON J.The economic impacts of foot and mouth disease——What are they,how big are they and where do they occur[J].Preventive Veterinary Medicine,2013,112(3-4):161-173.
[3] DOMINGO E,ESCARMíS C,BARANOWSKI E,et al.Evolution of foot-and-mouth disease virus[J].Virus Research,2003,91(1):47-63.
[4] DIAZ-SAN SEGUNDO F,MEDINA G N,STENFELDT C,et al.Foot-and-mouth disease vaccines[J].Veterinary Microbiology,2017,206:102-112.
[5] LEE S Y,LEE Y J,KIM R H,et al.Rapid engineering of foot-and-mouth disease vaccine and challenge viruses[J].Journal of Virology,2017,91(16):e00155-17.
[6] FRY E E,STUART D I,ROWLANDS D J.The structure of foot-and-mouth disease virus[J].Current Topics in Microbiology & Immunology,2005,288:71-101.
[7] HARANDI A M,MEDAGLINI D,SHATTOCK R J.Vaccine adjuvants:A priority for vaccine research[J]. Vaccine,2010,28(12):2363-2366.
[8] PUCKETTE M,CLARK B A,SMITH J D,et al.Foot-and-mouth disease virus 3C protease mutant L127P:Implications for FMD vaccine development[J].Journal of Virology,2017,91(22):e00924-17.
[9] KITCHING R P.Global epidemiology and prospects for control of foot-and-mouth disease[J].Current Topics in Microbiology & Immunology,2005,288(25):133-148.
[10] DONG Y M,CAI J C,CHEN H T,et al.Protection of a novel epitope-RNA VLP double-effective VLP vaccine for foot-and-mouth disease[J].Antiviral Research,2016,134:108-116.
[11] WANG D,FANG L,LIU L,et al.Foot-and-mouth disease virus (FMDV) leader proteinase negatively regulates the porcine interferon-λ1 pathway[J].Molecular Immunology,2011,49(1-2):407-412.
[12] OSTROWSKI M,VERMEULEN M,ZABAL O,et al.Impairment of thymus-dependent responses by murine dendritic cells infected with foot-and-mouth disease virus[J].Journal of Immunology,2005,175(6):3971-3979.
[13] PACHECO J M,BRUM M C,MORAES M P,et al.Rapid protection of cattle from direct challenge with foot-and-mouth disease virus (FMDV) by a single inoculation with an adenovirus-vectored FMDV subunit vaccine[J].Virology,2005,337(2):205-209.
[14] SANTOS T D L,BOTTON S D A,WEIBLEN R,et al.The leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mRNA and blocks the host innate immune response[J].Journal of Virology,2006,80(4):1906-1914.
[15] WANG D,FANG L,LI P,et al.The leader proteinase of foot-and-mouth disease virus negatively regulates the type Ⅰ interferon pathway by acting as a viral deubiquitinase[J].Journal of Virology,2011,85(8):3758-3766.
[16] 中华人民共和国农业农村部公告第2078号文件[Z].2014. Ministry of Agriculture and Rural Affairs of the People’s Republic of China.Announcement No.2078[Z].2014.(in Chinese)
[17] JIANG S,BAI X,LI P,et al.Influence of foot-and-mouth disease virus O/CHN/Mya98/33-P strain leader protein on viral replication and host innate immunity[J]. Viral Immunology,2015,28(7):360-366.
[18] PARK J N,KO M K,KIM R H,et al.Construction of stabilized and tagged foot-and-mouth disease virus[J].Journal of Virological Methods,2016,237:187-191.
[19] WANG D,FANG L,LUO R,et al.Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced typeⅠ interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels[J].Biochemical & Biophysical Research Communications,2010,399(1):72-78.
[20] PULIDO M R,SáIZ M.Molecular mechanisms of foot-and-mouth disease virus targeting the host antiviral response[J].Frontiers in Cellular & Infection Microbiology,2017,7:252-261.
[21] STENFELDT C,PACHECO J M,RODRIGUEZ L L,et al.Infection dynamics of foot-and-mouth disease virus in pigs using two novel simulated-natural inoculation methods[J].Research in Veterinary Science,2014,96(2):396-405.
[22] MORAES M P,SEGUNDO D S,DIAS C C,et al.Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response[J].Vaccine,2011,29(51):9431-9440.
[23] ROSAS M F,VIEIRA Y A,POSTIGO R,et al.Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus[J].Virology,2008,380(1):34-45.
[24] ROBIOLO B,TORRE J L,MARADEI E,et al.Confidence in indirect assessment of foot-and-mouth disease vaccine potency and vaccine matching carried out by liquid phase ELISA and virus neutralization tests[J].Vaccine,2010,28(38):6235-6241.
[25] CAO Y,DONG L,FU Y,et al.Rational design and efficacy of a multi-epitope recombinant protein vaccine against foot-and-mouth disease virus serotype A in pigs[J].Antiviral Research,2017,140:133-141.
[26] PARK M E,YOU S H,LEE S Y,et al.Immune responses in pigs and cattle vaccinated with half-volume foot-and-mouth disease vaccine[J]. Journal of Veterinary Science,2017,18(S1):323-331.
[27] BISWAL J K,BISHT P,MOHAPATRA J K,et al.Application of a recombinant capsid polyprotein (P1) expressed in a prokaryotic system to detect antibodies against foot-and-mouth disease virus serotype O[J].Journal of Virological Methods,2015,215-216:45-51.
[28] LI X,WANG J,LIU J,et al.Engagement of soluble resistance-related calcium binding protein (sorcin) with foot-and-mouth disease virus (FMDV) VP1 inhibits type Ⅰ interferon response in cells[J].Veterinary Microbiology,2013,166(1-2):35-46.
[29] 郭慧琛,孙世琪,云涛,等.口蹄疫病毒3D蛋白对DNA疫苗免疫效果的影响[J].中国兽医科学,2004,34(3):12-17. GUO H C,SUN S Q,YUN T,et al.The foot-and-mouth disease 3D protein effect on immune response of DNA vaccine[J].Chinese Veterinary Science,2004,34(3):12-17.(in Chinese)
[2] KNIGHT-JONES T J D,RUSHTON J.The economic impacts of foot and mouth disease——What are they,how big are they and where do they occur[J].Preventive Veterinary Medicine,2013,112(3-4):161-173.
[3] DOMINGO E,ESCARMíS C,BARANOWSKI E,et al.Evolution of foot-and-mouth disease virus[J].Virus Research,2003,91(1):47-63.
[4] DIAZ-SAN SEGUNDO F,MEDINA G N,STENFELDT C,et al.Foot-and-mouth disease vaccines[J].Veterinary Microbiology,2017,206:102-112.
[5] LEE S Y,LEE Y J,KIM R H,et al.Rapid engineering of foot-and-mouth disease vaccine and challenge viruses[J].Journal of Virology,2017,91(16):e00155-17.
[6] FRY E E,STUART D I,ROWLANDS D J.The structure of foot-and-mouth disease virus[J].Current Topics in Microbiology & Immunology,2005,288:71-101.
[7] HARANDI A M,MEDAGLINI D,SHATTOCK R J.Vaccine adjuvants:A priority for vaccine research[J]. Vaccine,2010,28(12):2363-2366.
[8] PUCKETTE M,CLARK B A,SMITH J D,et al.Foot-and-mouth disease virus 3C protease mutant L127P:Implications for FMD vaccine development[J].Journal of Virology,2017,91(22):e00924-17.
[9] KITCHING R P.Global epidemiology and prospects for control of foot-and-mouth disease[J].Current Topics in Microbiology & Immunology,2005,288(25):133-148.
[10] DONG Y M,CAI J C,CHEN H T,et al.Protection of a novel epitope-RNA VLP double-effective VLP vaccine for foot-and-mouth disease[J].Antiviral Research,2016,134:108-116.
[11] WANG D,FANG L,LIU L,et al.Foot-and-mouth disease virus (FMDV) leader proteinase negatively regulates the porcine interferon-λ1 pathway[J].Molecular Immunology,2011,49(1-2):407-412.
[12] OSTROWSKI M,VERMEULEN M,ZABAL O,et al.Impairment of thymus-dependent responses by murine dendritic cells infected with foot-and-mouth disease virus[J].Journal of Immunology,2005,175(6):3971-3979.
[13] PACHECO J M,BRUM M C,MORAES M P,et al.Rapid protection of cattle from direct challenge with foot-and-mouth disease virus (FMDV) by a single inoculation with an adenovirus-vectored FMDV subunit vaccine[J].Virology,2005,337(2):205-209.
[14] SANTOS T D L,BOTTON S D A,WEIBLEN R,et al.The leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mRNA and blocks the host innate immune response[J].Journal of Virology,2006,80(4):1906-1914.
[15] WANG D,FANG L,LI P,et al.The leader proteinase of foot-and-mouth disease virus negatively regulates the type Ⅰ interferon pathway by acting as a viral deubiquitinase[J].Journal of Virology,2011,85(8):3758-3766.
[16] 中华人民共和国农业农村部公告第2078号文件[Z].2014. Ministry of Agriculture and Rural Affairs of the People’s Republic of China.Announcement No.2078[Z].2014.(in Chinese)
[17] JIANG S,BAI X,LI P,et al.Influence of foot-and-mouth disease virus O/CHN/Mya98/33-P strain leader protein on viral replication and host innate immunity[J]. Viral Immunology,2015,28(7):360-366.
[18] PARK J N,KO M K,KIM R H,et al.Construction of stabilized and tagged foot-and-mouth disease virus[J].Journal of Virological Methods,2016,237:187-191.
[19] WANG D,FANG L,LUO R,et al.Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced typeⅠ interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels[J].Biochemical & Biophysical Research Communications,2010,399(1):72-78.
[20] PULIDO M R,SáIZ M.Molecular mechanisms of foot-and-mouth disease virus targeting the host antiviral response[J].Frontiers in Cellular & Infection Microbiology,2017,7:252-261.
[21] STENFELDT C,PACHECO J M,RODRIGUEZ L L,et al.Infection dynamics of foot-and-mouth disease virus in pigs using two novel simulated-natural inoculation methods[J].Research in Veterinary Science,2014,96(2):396-405.
[22] MORAES M P,SEGUNDO D S,DIAS C C,et al.Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response[J].Vaccine,2011,29(51):9431-9440.
[23] ROSAS M F,VIEIRA Y A,POSTIGO R,et al.Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus[J].Virology,2008,380(1):34-45.
[24] ROBIOLO B,TORRE J L,MARADEI E,et al.Confidence in indirect assessment of foot-and-mouth disease vaccine potency and vaccine matching carried out by liquid phase ELISA and virus neutralization tests[J].Vaccine,2010,28(38):6235-6241.
[25] CAO Y,DONG L,FU Y,et al.Rational design and efficacy of a multi-epitope recombinant protein vaccine against foot-and-mouth disease virus serotype A in pigs[J].Antiviral Research,2017,140:133-141.
[26] PARK M E,YOU S H,LEE S Y,et al.Immune responses in pigs and cattle vaccinated with half-volume foot-and-mouth disease vaccine[J]. Journal of Veterinary Science,2017,18(S1):323-331.
[27] BISWAL J K,BISHT P,MOHAPATRA J K,et al.Application of a recombinant capsid polyprotein (P1) expressed in a prokaryotic system to detect antibodies against foot-and-mouth disease virus serotype O[J].Journal of Virological Methods,2015,215-216:45-51.
[28] LI X,WANG J,LIU J,et al.Engagement of soluble resistance-related calcium binding protein (sorcin) with foot-and-mouth disease virus (FMDV) VP1 inhibits type Ⅰ interferon response in cells[J].Veterinary Microbiology,2013,166(1-2):35-46.
[29] 郭慧琛,孙世琪,云涛,等.口蹄疫病毒3D蛋白对DNA疫苗免疫效果的影响[J].中国兽医科学,2004,34(3):12-17. GUO H C,SUN S Q,YUN T,et al.The foot-and-mouth disease 3D protein effect on immune response of DNA vaccine[J].Chinese Veterinary Science,2004,34(3):12-17.(in Chinese)