中华蜜蜂囊状幼虫病病毒单克隆抗体的制备与鉴定
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摘要
为了制备中华蜜蜂囊状幼虫病病毒(CSBV)单克隆抗体,本实验利用纯化的中华蜜蜂囊状幼虫病毒(CSBV)免疫Balb/c小鼠,经过3次免疫后,小鼠断尾采血测其血清效价,选择效价高于1∶80 000的小鼠,在细胞融合前3~4d再加强免疫一次。取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,用间接ELISA筛选阳性细胞株并进行3次亚克隆后注射入Balb/c小鼠(Mus musculus)腹腔制备单抗腹水。共获得9株稳定分泌抗CSBV的单克隆抗体,命名为10A1、5B10、5A5、5H2、11D7、9A5、1D3、3C10、10C4,效价都在1∶16 000以上,经间接ELISA实验检测表明,具有较好的特异性。在此基础上,将9株腹水单抗分别与CSBV互作后,接种于2~3日龄中华蜜蜂幼虫,观察幼虫死亡率,研究单克隆抗体中和CSBV病毒能力,结果表明筛选到三株单克隆抗体(10A1、5A5、9A5)对CSBV有中和作用,为CSBV的防治研究奠定了基础。
We wished to prepare and identify monoclonal antibodies against the Chinese honeycomb cystic larvae virus(CSBV).First,the Chinese sacbrood bee virus was used thrice to immunize Balb/c mice.Then,we collected blood from tail veins to test their serum titer;We selected mice with a titer higher than1∶80,000.We immunized mice again 3~4 days before cell fusion.We undertook cell fusion between the splenocytes of immunized mice and SP2/0 myeloma cells.We applied an indirect enzyme-linked immunosorbent assay(ELISA)to screen positive cell strains after cell fusion.After sub-cloning these screened positive cell strains thrice,we injected them into the abdominal cavity of Balb/c mice to prepare ascites monoclonal antibodies.We obtained nine monoclonal antibodies that secreted anti-CSBV steadily and named them 10 A1,5 B10,5 A5,5 H2,11 D7,9 A5,1 D3,3 C10 and 10 C4.The titers of these nine ascites monoclonal antibodies were higher than 1∶16,000.Results of the indirect ELISA suggested that these nine ascites monoclonal antibodies had good specificity.On the basis of that result,these nine ascites monoclonal antibodies were interacted with CSBV,and then inoculated to 2~3-day-old Chinese bee larvae.The mortality of Chinese bee larvae was observed to study the neutralizing capacity of monoclonal antibodies to CSBV.Of the nine ascites monoclonal antibodies screened,three(10 A1,5 A5,9 A5)had a neutralizing effect no CSBV.The present study lays a foundation for the sdudy of the prevention and treatment of CSBV.
We wished to prepare and identify monoclonal antibodies against the Chinese honeycomb cystic larvae virus(CSBV).First,the Chinese sacbrood bee virus was used thrice to immunize Balb/c mice.Then,we collected blood from tail veins to test their serum titer;We selected mice with a titer higher than1∶80,000.We immunized mice again 3~4 days before cell fusion.We undertook cell fusion between the splenocytes of immunized mice and SP2/0 myeloma cells.We applied an indirect enzyme-linked immunosorbent assay(ELISA)to screen positive cell strains after cell fusion.After sub-cloning these screened positive cell strains thrice,we injected them into the abdominal cavity of Balb/c mice to prepare ascites monoclonal antibodies.We obtained nine monoclonal antibodies that secreted anti-CSBV steadily and named them 10 A1,5 B10,5 A5,5 H2,11 D7,9 A5,1 D3,3 C10 and 10 C4.The titers of these nine ascites monoclonal antibodies were higher than 1∶16,000.Results of the indirect ELISA suggested that these nine ascites monoclonal antibodies had good specificity.On the basis of that result,these nine ascites monoclonal antibodies were interacted with CSBV,and then inoculated to 2~3-day-old Chinese bee larvae.The mortality of Chinese bee larvae was observed to study the neutralizing capacity of monoclonal antibodies to CSBV.Of the nine ascites monoclonal antibodies screened,three(10 A1,5 A5,9 A5)had a neutralizing effect no CSBV.The present study lays a foundation for the sdudy of the prevention and treatment of CSBV.
引文
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[13]李芳兵,费东亮,张皓淳,胡影,魏东,张鹤,岳金金,马鸣潇,曲祖乙.中蜂囊状幼虫病毒结构蛋白VP2的密码子优化表达及其免疫原性研究[J].病毒学报,2017,33(2):231-237.
[14]王凌凤,杨涛,孙恩成,耿宏伟,秦永丽,赵晶,蔡绪禹,刘霓红,李文京,吴东来.蓝舌病病毒17型VP2蛋白单克隆抗体的制备及其抗原表位的鉴定[J].中国预防兽医学报,2011,33(6):465-470.
[15]姬希文,闫丽萍,颜丕熙,李国新,张七斤,李泽君.鸭坦布苏病毒抗体间接ELISA检测方法的建立[J].中国预防兽医学报,2011,33(8):630-634.
[16]孔君,刘箐,韩跃武,王天昌,刘金芝.单克隆抗体制备技术的最新进展及应用前景[J].免疫学杂志,2011,27(2):170-173.
[17]李阿茜,刘林,张硕,李川,张全福,梁米芳,李德新.抗发热伴血小板减少综合征病毒结构蛋白单克隆抗体的制备和功能分析[J].病毒学报,2015,31(1):18-23.
[18]陶嵘,倪振华,阳圣,刘冲,朱敏,沈江帆,涂少华.抗PSMA膜外域单克隆抗体的制备及初步应用[J].中国免疫学杂志,2016,32(2):205-209.
[19]贾晓晖,周芳,赵霞,李婷,贾天军.沙眼衣原体质粒蛋白pgp3单克隆抗体制备及鉴定[J].中国免疫学杂志,2015,31(5):659-662.
[20]李姝璇,杨立生,侯汪衡,赵欢,万俊凯,陈梦媛,何水珍,程通,夏宁邵.埃可病毒25型新型中和实验方法的建立与应用[J].生物技术通讯,2016,27(1):52-57.
[2]Liu X,Zhang Y,Yan X.Prevention of Chinese sacbrood virus infection in apis cerana using RNA interference[J].Curr Microbiol,2010,61(5):422-428.
[3]Khaliunaa Tsevegmid,Peter Neumann,Orlando Ya1ez.The honey bee pathosphere of mongolia:European viruses in Central Asia[J/OL].PLoS One,2016,11(3):e0151164.
[4]Ian Cavigli,Katie F.Daughenbaugh,Madison Martin,Michael Lerch,Katie Banner,Emma Garcia,Laura M.Brutscher,Michelle L.Flenniken.pathogen prevalence and abundance in honey bee colonies involved in almond pollination[J].Apidologie,2016,47:251-266.
[5]Suresh D.Desai,Robert W.Currie.Effects of wintering environment and parasite-pathogen interactions on honey bee colony loss in north temperate regions[J/OL].PLoS One,2016,11(7):e0159615.
[6]Ma Mingxiao,Li Ming,Cheng Jian,Yang Song,Wang Shude,and Li Pengfei.Molecular and biological characterization of Chinese sacbrood virus LN isolate[J].Comp Funct Genomics,2011,2011:409386.
[7]Ying Hu,Dongliang Fei,Lili Jiang,DongWei,Fangbing Li,Qingyun Diao,Mingxiao Ma.A comparison of biological characteristics of three strains of Chinese sacbrood virus in Apis cerana[J].Scientific Reports,2016,6:37424.
[8]马鸣潇,张大利,王立辛,袁小波,宋英今,和绍禹,费东亮,韩喜彬,徐晓丽.一种卵黄抗体的制备方法、用该方法制得的卵黄抗体及其应用[P].辽宁:CN102504023A,2012-06-20.
[9]马鸣潇,张大利,韩喜斌,袁小波,樊琼,郑洪玲,郑林,李慧.中蜂囊状幼虫病毒抗体的ELISA检测方法[P].辽宁:CN103760344A,2014-04-30.
[10]Mingxiao Ma,Chen Ma,Ming Li,Shuquan Wang,Song Yang,Shudue Wang.Loop-mediated isothermal amplification for rapid detection of Chinese sacbrood virus[J].J Virol Methods,2011,176(1-2):115-119.
[11]冯建勋,张勤奋,马泽微,张景强,黄文忠,张学峰.中蜂囊状幼虫病毒的纯化、结晶与结构研究[J].电子显微学报,1998,(4):74-75.
[12]Mingxiao Ma.New insights of sacbrood virus[J].Virol Sin,2014,29(6):410-413.
[13]李芳兵,费东亮,张皓淳,胡影,魏东,张鹤,岳金金,马鸣潇,曲祖乙.中蜂囊状幼虫病毒结构蛋白VP2的密码子优化表达及其免疫原性研究[J].病毒学报,2017,33(2):231-237.
[14]王凌凤,杨涛,孙恩成,耿宏伟,秦永丽,赵晶,蔡绪禹,刘霓红,李文京,吴东来.蓝舌病病毒17型VP2蛋白单克隆抗体的制备及其抗原表位的鉴定[J].中国预防兽医学报,2011,33(6):465-470.
[15]姬希文,闫丽萍,颜丕熙,李国新,张七斤,李泽君.鸭坦布苏病毒抗体间接ELISA检测方法的建立[J].中国预防兽医学报,2011,33(8):630-634.
[16]孔君,刘箐,韩跃武,王天昌,刘金芝.单克隆抗体制备技术的最新进展及应用前景[J].免疫学杂志,2011,27(2):170-173.
[17]李阿茜,刘林,张硕,李川,张全福,梁米芳,李德新.抗发热伴血小板减少综合征病毒结构蛋白单克隆抗体的制备和功能分析[J].病毒学报,2015,31(1):18-23.
[18]陶嵘,倪振华,阳圣,刘冲,朱敏,沈江帆,涂少华.抗PSMA膜外域单克隆抗体的制备及初步应用[J].中国免疫学杂志,2016,32(2):205-209.
[19]贾晓晖,周芳,赵霞,李婷,贾天军.沙眼衣原体质粒蛋白pgp3单克隆抗体制备及鉴定[J].中国免疫学杂志,2015,31(5):659-662.
[20]李姝璇,杨立生,侯汪衡,赵欢,万俊凯,陈梦媛,何水珍,程通,夏宁邵.埃可病毒25型新型中和实验方法的建立与应用[J].生物技术通讯,2016,27(1):52-57.