兔抗O型口蹄疫VLPs酶标抗体的制备及初步应用
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摘要
为制备兔抗O型口蹄疫病毒样颗粒(VLPs)的酶标抗体,进一步建立O型口蹄疫病毒的ELISA检测方法,通过原核表达、体外纯化和组装O型口蹄疫VLPs,免疫家兔制备兔抗O型口蹄疫VLPs高免血清,并采用亲和层析法纯化IgG,改良过碘酸钠法制备兔抗O型口蹄疫VLPs的酶标抗体(rabbit anti-type O foot-and-mouth disease IgG-HRP)。结果显示,兔抗O型口蹄疫VLPs高免血清效价达1∶4 096,纯化后的IgG纯度达90%,效价达1∶512 000。表明获得了高纯度的兔抗O型口蹄疫VLP的酶标抗体,可用于O型口蹄疫的ELISA检测。
In order to prepare the rabbit anti-type O foot-and-mouth disease virus VLPs IgG-HRP,and further establish ELISA method for detecting type O foot-and-mouth disease virus,the VLPs of O type FMDV was prokaryotially expressed,purified and assembled in vitro.Immunized rabbits with type O FMDV VLPs were used to prepare rabbit anti-O-type FMDV VLPs hyper-immune serum.To purify IgG by affinity chromatography,and prepare the rabbit anti-type O FMDV VLPs IgG-HRP by sodium periodate oxidation method.The titer for rabbit anti-O-type FMDV VLPs hyper-immune serum was up to 1∶4 096,and the rabbit anti-type O FMDV VLPs IgG was purified up to 90%,the titer of IgG was up to 1∶512 000.The result showed that high purity rabbit anti-type O FMDV VLPs IgG-HRP was prepared,which has the potential to be used for ELISA detection of type O FMDV.
In order to prepare the rabbit anti-type O foot-and-mouth disease virus VLPs IgG-HRP,and further establish ELISA method for detecting type O foot-and-mouth disease virus,the VLPs of O type FMDV was prokaryotially expressed,purified and assembled in vitro.Immunized rabbits with type O FMDV VLPs were used to prepare rabbit anti-O-type FMDV VLPs hyper-immune serum.To purify IgG by affinity chromatography,and prepare the rabbit anti-type O FMDV VLPs IgG-HRP by sodium periodate oxidation method.The titer for rabbit anti-O-type FMDV VLPs hyper-immune serum was up to 1∶4 096,and the rabbit anti-type O FMDV VLPs IgG was purified up to 90%,the titer of IgG was up to 1∶512 000.The result showed that high purity rabbit anti-type O FMDV VLPs IgG-HRP was prepared,which has the potential to be used for ELISA detection of type O FMDV.
引文
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[14]聂传朋,尚亚南.从血清中提取IgG免疫球蛋白[J].阜阳师范学院学报:自然科学版,2002(2):16-17.
[15]夏凯,李建科,于振.酸土环脂芽孢杆菌的鼠源多克隆抗体制备及纯化[J].食品工业科技,2012,33(15):162-164,168.
[16] Sjoquist J,Movitz J,Johansson I B,et al.Localization of protein A in the bacteria[J].Eur J Biochem,1972(30):190-194.
[17] Lindmark R,Thoren-Tolling K,Sjoquist J.Binding of immunoglobulin to protein A and immunoglobulin levels in mammalian sera[J].J Immunol Mothods,1983(62):1-13.
[18] Brito A I,Acosta Bas C,Rodriguez M,et al.Monoclonal antibodies to the recombinant protein TmpA of the Treponema pallidum[J].HybridomaHybridomics,2003,22(6):393-396.
[19]顾静,杨锦媛,周庆.检测PAIg酶标抗体制备中的影响因素探讨[J].南通医学院学报,1994(2):248-249.
[2] Jamal S M,Belsham G J.Foot-and-mouth disease:past,present and future[J].Vet Res,2013,44(44):1-14.
[3]谢庆阁.口蹄疫[M].北京:中国农业出版社,2004:1-15.
[4]吕晓丽,姚晓韵,何奇松,等.两种O型口蹄疫ELISA检测试剂盒的对比[J].中国动物检疫,2016,33(3):71-73.
[5] Grgacic E V,Anderson D A.Virus-like particles:passport to immune recognition[J].Methods,2006,40(1):60-65
[6] Logan D,Abu-Ghazaleh R,Blakemore W,et al.Structure of a major immunogenic site on foot-and-mouth disease virus[J].Nature,1993,362(6420):566-568.
[7]蔡宝祥.家畜传染病学[M].4版.北京:中国农业出版社,2006:155-158.
[8] Chitray M,Beer T A P D,Vosloo W.Genetic heterogeneity in the leader and P1-coding regions of foot-and-mouth disease virus serotypes A and O in Africa[J].Arch Virol,2014,159(5):947-961.
[9] Vanlierop M J,Wagenaar J P,Vannoort J M.Sequence derived from the highly antigenic VP1region 140to 160of foot-andmouth disease virus do not prime for a bovine T-cell response against intact virus[J].J Virol,1995,69(7):4511-4514.
[10] Pattenden L K,Middelberg A P J,Niebert M,et al.Toward the preparative and large-scale precision manufacture of viruslike particles[J].Trends Biotechnol,2005,23(10):523-529.
[11] Lee C D,Yan Y P,Lianget S M,et al.Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli[J].J Biomed Sci,2009,16(1):1-7.
[12] Harlow E,David L.Antibody:A laboratory manual[M].New York:Cold Spring Harbor Laboratory,1988:284-300.
[13]张大川,付云洁,刘志国,等.不同纯化兔抗苏丹红ⅠIgG方法的比较[J].武汉工业学院学报,2011,30(2):5-7.
[14]聂传朋,尚亚南.从血清中提取IgG免疫球蛋白[J].阜阳师范学院学报:自然科学版,2002(2):16-17.
[15]夏凯,李建科,于振.酸土环脂芽孢杆菌的鼠源多克隆抗体制备及纯化[J].食品工业科技,2012,33(15):162-164,168.
[16] Sjoquist J,Movitz J,Johansson I B,et al.Localization of protein A in the bacteria[J].Eur J Biochem,1972(30):190-194.
[17] Lindmark R,Thoren-Tolling K,Sjoquist J.Binding of immunoglobulin to protein A and immunoglobulin levels in mammalian sera[J].J Immunol Mothods,1983(62):1-13.
[18] Brito A I,Acosta Bas C,Rodriguez M,et al.Monoclonal antibodies to the recombinant protein TmpA of the Treponema pallidum[J].HybridomaHybridomics,2003,22(6):393-396.
[19]顾静,杨锦媛,周庆.检测PAIg酶标抗体制备中的影响因素探讨[J].南通医学院学报,1994(2):248-249.