基因重排狂犬病病毒疫苗株免疫效果的初步研究
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摘要
试验探究了基因重排狂犬病病毒(RV)弱毒疫苗株能否刺激小鼠产生RV抗体免疫球蛋白G(IgG),比较了其不同免疫方式、不同免疫剂量对小鼠抗体水平的影响及安全性评估。选取28只体重(20±3)g、35日龄左右的昆明系小白鼠,随机分为7组:第1组为低剂量口服组:复合佐剂50μL+病毒液50μL;第2组为中等剂量口服组:复合佐剂150μL+病毒液150μL;第3组为高剂量口服组:复合佐剂300μL+病毒液300μL;第4组为低剂量注射组:复合佐剂50μL+病毒液50μL;第5组为中等剂量注射组:复合佐剂150μL+病毒液150μL;第6组为高剂量注射组:复合佐剂300μL+病毒液300μL;第7组为口服对照组:复合佐剂300μL+SRV9亲本毒株300μL,分别于0、7、14d进行免疫,采用ELISA定量检测试剂盒对各免疫组小鼠的血清IgG水平进行检测,免疫期结束后取各免疫组小鼠的肝脏、肾脏、肺脏、脑组织制作病理组织切片对疫苗的安全性进行评估。结果显示,各免疫组均产生了RV抗体IgG;肌内注射免疫产生的抗体速度较口服免疫快,但肌内注射免疫会导致小鼠发病死亡;600μL口服免疫组产生的抗体水平显著高于100、300μL口服免疫组及亲本毒株SRV9对照组(P<0.05),说明适当加大口服免疫剂量会使口服免疫组产生的抗体水平增加,并且不会引起不良反应。病理组织学检查发现,各口服免疫组小鼠的肝脏、肾脏、肺脏、脑组织的形态结构较为正常,未发生病变,表明基因重排RV疫苗株口服免疫副作用小、安全性较好。本试验结果为研制新型口服疫苗开辟了新的方向。
To explore whether gene rearrangement rabies virus(RV)vaccine could stimulate the production of RV antibody IgG in mice,and compare the IgG level and safety performance in different immune modes and different immune doses of attenuated RV vaccine,28 35-day-old Kunming mice with(20±3)g were chosen and assigned to 7groups:Group 1(low dose oral group,50μL compound adjuvant+50μL RV),group 2(middle dose oral group,150μL compound adjuvant+150μL RV),group 3(high dose oral group,300μL compound adjuvant+300μL RV),group 4(low dose injection group,50μL compound adjuvant+50μL RV),group 5(middle dose injection group,150μL compound adjuvant+150μL RV),group 6(high dose injection group,300μL compound adjuvant+300μL RV)and group 7(oral control group,300μL compound ad-juvant+300μL SRV9strain).Immunisation was performed at 0,7and 14 d,respectively,and the IgG level of mice in each immune group was detected by ELISA kit,and the liver,kidney,lung,brain tissues of the immune groups were collected to evaluate the safety of the vaccine at the end of immunization test.The results showed that IgG was produced in each immune group.The rate of IgG produced by intramuscular injection was higher than that of oral immunization,but intramuscular vaccination could lead to death of mice.The IgG level produced by 600μL oral immunization group was significantly higher than 100 and 300μL oral immune groups and parental strains SRV9 control group(P<0.05),which indicated that increasing oral immunization dose would increase the level of antibody produced by oral immune and would not cause adverse reactions.Histopathological examination found that the liver,kidney,lung and brain tissues of each oral immune group were normal and did not see pathological changes,indicating that the gene rearrangement RV had less side effects and had better safety.It provided a new direction for the development of a new type of RV attenuated oral vaccine.
To explore whether gene rearrangement rabies virus(RV)vaccine could stimulate the production of RV antibody IgG in mice,and compare the IgG level and safety performance in different immune modes and different immune doses of attenuated RV vaccine,28 35-day-old Kunming mice with(20±3)g were chosen and assigned to 7groups:Group 1(low dose oral group,50μL compound adjuvant+50μL RV),group 2(middle dose oral group,150μL compound adjuvant+150μL RV),group 3(high dose oral group,300μL compound adjuvant+300μL RV),group 4(low dose injection group,50μL compound adjuvant+50μL RV),group 5(middle dose injection group,150μL compound adjuvant+150μL RV),group 6(high dose injection group,300μL compound adjuvant+300μL RV)and group 7(oral control group,300μL compound ad-juvant+300μL SRV9strain).Immunisation was performed at 0,7and 14 d,respectively,and the IgG level of mice in each immune group was detected by ELISA kit,and the liver,kidney,lung,brain tissues of the immune groups were collected to evaluate the safety of the vaccine at the end of immunization test.The results showed that IgG was produced in each immune group.The rate of IgG produced by intramuscular injection was higher than that of oral immunization,but intramuscular vaccination could lead to death of mice.The IgG level produced by 600μL oral immunization group was significantly higher than 100 and 300μL oral immune groups and parental strains SRV9 control group(P<0.05),which indicated that increasing oral immunization dose would increase the level of antibody produced by oral immune and would not cause adverse reactions.Histopathological examination found that the liver,kidney,lung and brain tissues of each oral immune group were normal and did not see pathological changes,indicating that the gene rearrangement RV had less side effects and had better safety.It provided a new direction for the development of a new type of RV attenuated oral vaccine.
引文
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[19]刘澜,李士成,陈小云,等.重组表达3G蛋白狂犬病病毒株的构建及重组病毒相关特性的研究[J].中国兽医科学,2016,46(3):326-331.LIU L,LI S C,CHEN X Y,et al.Construction and properties of a recombinant rabies virus expressing triple G proteins[J].Chinese Veterinary Science,2016,46(3):326-331.(in Chinese)
[20]HUANG Y,CHEN Z,HUANG J,et al.Parainfluenza virus 5expressing the G protein of rabies virus protects mice after rabies virus infection[J].Journal of Virology,2015,89(6):3427-3429.
[21]赵静.表达犬瘟热病毒H基因重组狂犬病病毒生物学特性的研究[D].广州:华南农业大学,2016.ZHAO J.Construction of recombinant pseudorabies virus expressing canine distemper virus H gene and analysis on its biological characters[D].Guangzhou:South China Agricultural University,2016.(in Chinese)
[22]张亚静.表达IL-21重组狂犬病病毒免疫原性研究[D].武汉:华中农业大学,2017.ZHANG Y J.The immunogenicity investigation of recombinant rabies virus expressing IL-21[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[2]LIU D.Molecular Detection of Animal Viral Pathogens[M].Florida:CRC Press,2016.
[3]CONZELMANN K K.Reverse Genetics of Mononegavirales:The Rabies Virus Paradigm[M].Japan:Springer,2013.
[4]孙雨,马世春,王晓英,等.狂犬病病毒流行病学特征与实验室诊断技术的研究进展[J].中国畜牧兽医,2015,42(2):487-492.SUN Y,MA S C,WANG X Y,et al.Research progress on the epidemiological characteristics and diagnosis technology of rabies virus[J].China Animal Husbandry&Veterinarian Medicine,2015,42(2):487-492.(in Chinese)
[5]FISHER C R,STREICKER D G,SCHNELL M J.The spread and evolution of rabies virus:Conquering new frontiers[J].Nature Reviews Microbiology,2018,16(4):241-255.
[6]OSANAI Y,SHIMIZU T,MORI T,et al.Rabies virus-mediated oligodendrocyte labeling reveals a single oligodendrocyte myelinates axons from distinct brain regions[J].Glia,2017,65(1):93-105.
[7]JARCHUM I.Neuroscience:On-demand rabies[J].Nature Methods,2017,14(9):836.
[8]FENNER F J,BACHMANN P A,GIBBS E P J.Veterinary Virology[M].Carolina:Academic Press,2014.
[9]HIDAKA Y,LIM C K,TAKAYAMA-ITO M,et al.Segmentation of the rabies virus genome[J].Virus Research,2018,252:68-75.
[10]STARODUBOVA E S,KUZMENKO Y V,PANKO-VA E O,et al.A DNA construct that encodes the rabies virus consensus glycoprotein with a proteasome degradation signal induces antibody production with IgG2Asubtype predominance[J].Molecular Biology,2018,52(3):453-457.
[11]古丽麦拉·卡日,简子健,夏婷婷,等.狂犬病口服疫苗SRV_9毒株基因序列的生物信息学分析[J].中国畜牧兽医,2015,42(6):1355-1361.GULIMELA·KARI,JIAN Z J,XIA T T,et al.Bioinformatics analysis of gene sequence of oral rabies vaccine SRV_9strain[J].China Animal Husbandry&Veterinarian Medicine,2015,42(6):1355-1361.(in Chinese)
[12]GHANEM A,CONZELMANNK K.Ggene-deficient single-round rabies viruses for neuronal circuit analysis[J].Virus Research,2016,216:41-54.
[13]ZIENIUS D,PRIDOTKAS G,MORKUNAS M,et al.Phylogenetic investigation of partial Ggene sequences in Lithuanian rabies virus field isolates[J].Veterinarski Arhiv,2016,86(1):23-34.
[14]陈晶,刘晓慧,艾军,等.糖蛋白基因重排至第二位对狂犬病病毒生物学特性的影响[A].2010全国狂犬病防控高层论坛论文集[C].2010.CHEN J,LIU X H,AI J,et al.Characterization and rescue of Ggene rearrangements virus[A].2010National High-level Forum on Rabies Prevention[C].2010.(in Chinese)
[15]DEVIATKIN A A,LUKASHEV A N.Recombination in the rabies virus and other lyssaviruses[J].Infection,Genetics and Evolution,2018,60:97-102.
[16]李明明.表达鼠CXCL13重组狂犬病病毒的构建及免疫原性的研究[D].武汉:华中农业大学,2016.LI M M.Construction and immunogenicity investigation of recombinant rabies virus expressing CXCL13[D].Wuhan:Huazhong Agricultural University,2016.(in Chinese)
[17]王丽娜.表达西尼罗病毒囊膜蛋白重组狂犬病病毒的构建与应用研究[D].长春:吉林农业大学,2016.WANG L N.Study on the construction and application of recombinant rabies virus expressing West Nile virus envelope protein[D].Changchun:Jilin Agricultural University,2016.(in Chinese)
[18]杨杰.表达Flt3L的重组狂犬病病毒的构建及其免疫原性的研究[D].武汉:华中农业大学,2016.YANG J.Construction and immunogenicity investigation of recombinant rabies virus expressing Flt3L[D].Wuhan:Huazhong Agricultural University,2016.(in Chinese)
[19]刘澜,李士成,陈小云,等.重组表达3G蛋白狂犬病病毒株的构建及重组病毒相关特性的研究[J].中国兽医科学,2016,46(3):326-331.LIU L,LI S C,CHEN X Y,et al.Construction and properties of a recombinant rabies virus expressing triple G proteins[J].Chinese Veterinary Science,2016,46(3):326-331.(in Chinese)
[20]HUANG Y,CHEN Z,HUANG J,et al.Parainfluenza virus 5expressing the G protein of rabies virus protects mice after rabies virus infection[J].Journal of Virology,2015,89(6):3427-3429.
[21]赵静.表达犬瘟热病毒H基因重组狂犬病病毒生物学特性的研究[D].广州:华南农业大学,2016.ZHAO J.Construction of recombinant pseudorabies virus expressing canine distemper virus H gene and analysis on its biological characters[D].Guangzhou:South China Agricultural University,2016.(in Chinese)
[22]张亚静.表达IL-21重组狂犬病病毒免疫原性研究[D].武汉:华中农业大学,2017.ZHANG Y J.The immunogenicity investigation of recombinant rabies virus expressing IL-21[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)