兔豆状囊尾蚴囊液蛋白的纯化与抗原性鉴定
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摘要
为了找出抗原性强、免疫原性好的兔豆状囊尾蚴抗原蛋白,本研究对兔豆状囊尾蚴的头节、体节和囊膜等结构性抗原与囊液中代谢性抗原进行了比较,发现囊液代谢性抗原的免疫效果优于结构性抗原。通过SDS-PAGE和微孔超滤分离法对囊液蛋白进行纯化,再用纯化的蛋白免疫小鼠,制备高免血清,并对免疫鼠的免疫器官进行病理学和免疫组织化学检查。结果证明用从囊液中纯化的63 000蛋白免疫效果最好。用纯化的63 000蛋白免疫小鼠可获得高效价抗血清,小鼠免疫器官活化,巨噬细胞、滤泡树突细胞、淋巴细胞和浆细胞等各种免疫细胞增多,用免疫组织化学染色,免疫器官中的CD4、CD8T细胞和CD20B细胞呈强阳性反应。总之,从囊液中分离纯化的63 000蛋白抗原性强、免疫原性,可用于对豆状囊尾蚴病的免疫学诊断。
To find out a protein of Cysticercus pisiformis with strong antigenicity and good immunogenicity antigen,the structure antigen of scolex,metamere and cyst membrane of Cysticercus pisiformis were compared with metabolic antigen of cyst fluid,the result showed that the immune effect of metabolic antigen was superior to structure antigen.SDS-PAGE and micropore ultrafiltration separation method were used to purify cyst fluid,purified cyst fluid protein was used to immune mice to get hyper-immune serum,immune organs of immune mice were examined by pathological and immunohistochemistry examination.As a result 63 000 protein purified from cyst fluid had a good immune effect.When 63 000 protein was used to immune mice,hyper-immune serum was obtained,immune organs of immune mice were in excitation,various kinds of immune cells,such as macrophages,follicular dendritic cells,lymphocytes and plasmocytes were increased in immune organs,immunohistochemistry displayed that there were strong positive reaction in CD4,CD8 Tcell and CD20 Bcell in immune organs of immune mice.In a word,63 000 protein purified from cyst fluid had a strong antigenicity and good immunogenicity antigen and could be used to diagnose Cysticercus pisiformis immunologically.
To find out a protein of Cysticercus pisiformis with strong antigenicity and good immunogenicity antigen,the structure antigen of scolex,metamere and cyst membrane of Cysticercus pisiformis were compared with metabolic antigen of cyst fluid,the result showed that the immune effect of metabolic antigen was superior to structure antigen.SDS-PAGE and micropore ultrafiltration separation method were used to purify cyst fluid,purified cyst fluid protein was used to immune mice to get hyper-immune serum,immune organs of immune mice were examined by pathological and immunohistochemistry examination.As a result 63 000 protein purified from cyst fluid had a good immune effect.When 63 000 protein was used to immune mice,hyper-immune serum was obtained,immune organs of immune mice were in excitation,various kinds of immune cells,such as macrophages,follicular dendritic cells,lymphocytes and plasmocytes were increased in immune organs,immunohistochemistry displayed that there were strong positive reaction in CD4,CD8 Tcell and CD20 Bcell in immune organs of immune mice.In a word,63 000 protein purified from cyst fluid had a strong antigenicity and good immunogenicity antigen and could be used to diagnose Cysticercus pisiformis immunologically.
引文
[1]BAGRADE G,KIRJUSINA M,VISMANIS K,et al.Helminths parasites of the wolf Canis lupus from Latvia[J].J Helminthol,2009,83(1):63-68.
[2]HALLORAN P,LAZOVICH D,PATTERSON R E,et al.Effect of health lifestyle pattern on dietary change[J].Am J Health Promot,2001,16:27-33.
[3]MIGUEL-ANGEL B A,AGUSTIN O,VIRGINIO A,et al.Changes in behavioural and physiological parameters associated with Taenia pisiformis infection in rabbits(Oryctolagus cuniculus)that may improve early detection of sick rabbits[J].World Rabbit Sci,2011,19(1):21-30.
[4]KRZYSZTOF S,RENATA R L,KLAUDIUSZ O S,et al.Occurrence of gastrointestinal parasites in slaughter rabbits[J].Parasitol Res,2014,113(1):59-64.
[5]王帅,韩利方,张高奎,等.屠宰兔感染豆状囊尾蚴的体内分布及卫生评价[J].动物医学进展,2009,30(9):11-15.
[6]周永学,杜爱芳,张雪娟,等.肉兔豆状囊尾蚴病的危害性研究[J].浙江农业科学,2008(3):372-373.
[7]张玲,郑国清,郑娟,等.商品兔豆状囊尾蚴病的流行及防治[J].中国兽医寄生虫病,2007,15(5):49-50.
[8]张金合,姜海清,姜诗旺.家兔豆状囊尾蚴病的防治[J].现代畜牧兽医,2009(9):38-39.
[9]吴跃华,张雄,刘小昌,等.商品兔豆状囊尾蚴病的流行及防治[J].兽医临床,2008,35(4):118.
[10]彭屹,林江蔚,郭志强.新西兰兔豆状囊尾蚴病的诊治[J].中国兽医杂志,2012,48(5):85.
[11]张守发,鞠玉琳,李学一,等.不同抗原在兔豆状囊尾蚴病血清学诊断中的研究[J].延边农学院学报,1994,16(2):98-102.
[12]张守发,鞠玉琳,池鹤松.诊断兔豆状囊尾蚴病的不同血清学检查方法的对比研究[J].延边农学院学报,1995,17(1):41-45.
[13]王晓霞,骆学农,孙晓林,等.豆状囊尾蚴人工感染家兔抗体水平的消长[J].中国兽医科学,2009,39(4):283-286.
[14]韩进欢,张小宇,张倩,等.豆状带绦虫不同发育阶段虫体的抗原特性分析[J].中国兽医科学,2012,42(12):1259-1263.
[15]卞晓锐,徐桐桐,单彩龙,等.淋病奈瑟菌WHO-A株Cas蛋白的表达和多克隆抗体制备[J].扬州大学学报(农业与生命科学版),2017,38(1)6-11.
[16]殷冬冬,白彩霞,唐井玉,等.Nectin-4基因的原核表达及多克隆抗体制备[J].扬州大学学报(农业与生命科学版),2016,37(3):6-10.
[17]孔繁瑶.家畜寄生虫学[M].北京:中国农业大学出版社,2008:146-147.
[18]BOYKO B,GEORGIE V,RODNEY A,et al.Littlewood.cestodes of small mammals:Taxonomy and life cycles[M].Micromammals and Macroparasites,2006:380-382.
[19]汪明.兽医寄生虫学[M].北京:中国农业出版社,2011:400-401.
[20]李德昌,张西臣,李建华.动物寄生虫学[M].北京:科学出版社,2010:400-402.
[21]BAGRADE G,KIRJUSINA M,VISMANIS K,et al.Helminth parasites of the wolf Canis lupus from Latvia[J].J Helminthology,2009,83(5):63-68.
[22]KYNGDON C T,GAUCI C G,ROKFE R A,et al.In vitro oncosphere-killing assays to determine immunity to the larvae of Taenia pisiformis,Taenia ovis,Taenia saginata,and Taenia solium[J].J Parasitol,2006,92(2):273-281.
[23]KENJIRO T,SHUNJI N,MASAHIRO M,et al.Abdominal cysticercosis in a cynomolgus monkey[J].Vet Par,2009,161(5):339-341.
[24]PAN Y Q,ZHAO Z S,CHEN J S,et al.Ultrastructure observation of cysticercus pisiformis and its function analysis[J].Ani Husban Fe Sci,2012,4(4):145-149.
[25]PAN Y Q,ZHANG L P,WANG S,et al.Ultrastructural changes of scolex on cysticercus pisiformis during relative rest and motion states[J].Agri Sci Tech,2014,15(1):111-115.
[26]潘耀谦,刘兴友,赵振升,等.豆状囊尾蚴的超微结构观察[J].畜牧兽医学报,2012,43(4):653-658.
[27]潘耀谦,刘兴友,银梅,等.豆状囊尾蚴头节压片与扫描电镜的比较观察[J].中国兽医学报,2012,32(3):441-446.
[2]HALLORAN P,LAZOVICH D,PATTERSON R E,et al.Effect of health lifestyle pattern on dietary change[J].Am J Health Promot,2001,16:27-33.
[3]MIGUEL-ANGEL B A,AGUSTIN O,VIRGINIO A,et al.Changes in behavioural and physiological parameters associated with Taenia pisiformis infection in rabbits(Oryctolagus cuniculus)that may improve early detection of sick rabbits[J].World Rabbit Sci,2011,19(1):21-30.
[4]KRZYSZTOF S,RENATA R L,KLAUDIUSZ O S,et al.Occurrence of gastrointestinal parasites in slaughter rabbits[J].Parasitol Res,2014,113(1):59-64.
[5]王帅,韩利方,张高奎,等.屠宰兔感染豆状囊尾蚴的体内分布及卫生评价[J].动物医学进展,2009,30(9):11-15.
[6]周永学,杜爱芳,张雪娟,等.肉兔豆状囊尾蚴病的危害性研究[J].浙江农业科学,2008(3):372-373.
[7]张玲,郑国清,郑娟,等.商品兔豆状囊尾蚴病的流行及防治[J].中国兽医寄生虫病,2007,15(5):49-50.
[8]张金合,姜海清,姜诗旺.家兔豆状囊尾蚴病的防治[J].现代畜牧兽医,2009(9):38-39.
[9]吴跃华,张雄,刘小昌,等.商品兔豆状囊尾蚴病的流行及防治[J].兽医临床,2008,35(4):118.
[10]彭屹,林江蔚,郭志强.新西兰兔豆状囊尾蚴病的诊治[J].中国兽医杂志,2012,48(5):85.
[11]张守发,鞠玉琳,李学一,等.不同抗原在兔豆状囊尾蚴病血清学诊断中的研究[J].延边农学院学报,1994,16(2):98-102.
[12]张守发,鞠玉琳,池鹤松.诊断兔豆状囊尾蚴病的不同血清学检查方法的对比研究[J].延边农学院学报,1995,17(1):41-45.
[13]王晓霞,骆学农,孙晓林,等.豆状囊尾蚴人工感染家兔抗体水平的消长[J].中国兽医科学,2009,39(4):283-286.
[14]韩进欢,张小宇,张倩,等.豆状带绦虫不同发育阶段虫体的抗原特性分析[J].中国兽医科学,2012,42(12):1259-1263.
[15]卞晓锐,徐桐桐,单彩龙,等.淋病奈瑟菌WHO-A株Cas蛋白的表达和多克隆抗体制备[J].扬州大学学报(农业与生命科学版),2017,38(1)6-11.
[16]殷冬冬,白彩霞,唐井玉,等.Nectin-4基因的原核表达及多克隆抗体制备[J].扬州大学学报(农业与生命科学版),2016,37(3):6-10.
[17]孔繁瑶.家畜寄生虫学[M].北京:中国农业大学出版社,2008:146-147.
[18]BOYKO B,GEORGIE V,RODNEY A,et al.Littlewood.cestodes of small mammals:Taxonomy and life cycles[M].Micromammals and Macroparasites,2006:380-382.
[19]汪明.兽医寄生虫学[M].北京:中国农业出版社,2011:400-401.
[20]李德昌,张西臣,李建华.动物寄生虫学[M].北京:科学出版社,2010:400-402.
[21]BAGRADE G,KIRJUSINA M,VISMANIS K,et al.Helminth parasites of the wolf Canis lupus from Latvia[J].J Helminthology,2009,83(5):63-68.
[22]KYNGDON C T,GAUCI C G,ROKFE R A,et al.In vitro oncosphere-killing assays to determine immunity to the larvae of Taenia pisiformis,Taenia ovis,Taenia saginata,and Taenia solium[J].J Parasitol,2006,92(2):273-281.
[23]KENJIRO T,SHUNJI N,MASAHIRO M,et al.Abdominal cysticercosis in a cynomolgus monkey[J].Vet Par,2009,161(5):339-341.
[24]PAN Y Q,ZHAO Z S,CHEN J S,et al.Ultrastructure observation of cysticercus pisiformis and its function analysis[J].Ani Husban Fe Sci,2012,4(4):145-149.
[25]PAN Y Q,ZHANG L P,WANG S,et al.Ultrastructural changes of scolex on cysticercus pisiformis during relative rest and motion states[J].Agri Sci Tech,2014,15(1):111-115.
[26]潘耀谦,刘兴友,赵振升,等.豆状囊尾蚴的超微结构观察[J].畜牧兽医学报,2012,43(4):653-658.
[27]潘耀谦,刘兴友,银梅,等.豆状囊尾蚴头节压片与扫描电镜的比较观察[J].中国兽医学报,2012,32(3):441-446.