双峰驼血清IgG亚型的分离纯化及多克隆抗体的制备
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摘要
双峰驼IgG亚型包含IgG1、IgG2和IgG3,其中IgG2和IgG3为重链抗体,在结构上与IgG1存在显著差异。为获取双峰驼血清中的IgG1、IgG2和IgG3,并分析其抗原特异性和抗体特异性,本文交替使用Protein A和Protein G亲和层析柱,对其分离纯化,并通过聚丙烯酰胺凝胶电泳进行鉴定;之后分别制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体,通过ELISA对制备的多克隆抗体的效价进行测定;最后应用Western blot评估这3个亚型多克隆抗体的特异性,进而对双峰驼血清中IgG1、IgG2和IgG3的抗原特异性进行分析。结果表明,应用Protein A和Protein G亲和层析柱成功分离纯化出双峰驼血清中的IgG1、IgG2和IgG3;并制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体效价均在1∶10 000以上,且所获得的多克隆抗体分别与IgG1、IgG2和IgG3之间均存在交叉反应,但兔抗双峰驼IgG1多克隆抗体较其它两个亚型多克隆抗体特异性低。结果证明,双峰驼IgG1、IgG2和IgG3均具有良好的免疫原性,三者结构虽存在显著差异,但其抗原特性类似。
IgGsubclasses of Bactrian camel is consist of IgG1,IgG2 and IgG3. Both IgG2 and IgG3 are the heavy chain antibodies( HCAbs),with structures that are obviously different from IgG1's. To obtain IgG1,IgG2 and IgG3 and analyze their antigen specificities,IgGsubclasses were isolated and purified from the Bactrian camel serum using Protein A and Protein G affinity chromatography columns,and were identified by polyacrylamide gel electrophoresis( SDS-PAGE). Then polyclonal antibodies to them were prepared respectively and their titers were determined by enzyme-linked immunosorbent assay( ELISA). Finally,their specificities were evaluated by Western blot,and the antigen-specificity of IgGsubclasses of Bactrian camel were further analyzed. The results showed that the IgG1,IgG2 and IgG3 of Bactrian camel are obtained successfully by the Protein A and Protein G affinity chromatography columns,the titers of the polyclonal antibodies of antiIgG1,IgG2,and IgG3 were all over 1∶ 10 000,and every polyclonal antibody had obvious cross-reactivity with IgG1,IgG2 and IgG3,but the polyclonal antibody specificity of anti-IgG1 was lower than those of anti-IgG2 and anti-IgG3. The results suggested that the immunogenicity of IgG1,IgG2 and IgG3 of Bactrian camel were all good and their specificities were all similar although their structures were obviously different.
IgGsubclasses of Bactrian camel is consist of IgG1,IgG2 and IgG3. Both IgG2 and IgG3 are the heavy chain antibodies( HCAbs),with structures that are obviously different from IgG1's. To obtain IgG1,IgG2 and IgG3 and analyze their antigen specificities,IgGsubclasses were isolated and purified from the Bactrian camel serum using Protein A and Protein G affinity chromatography columns,and were identified by polyacrylamide gel electrophoresis( SDS-PAGE). Then polyclonal antibodies to them were prepared respectively and their titers were determined by enzyme-linked immunosorbent assay( ELISA). Finally,their specificities were evaluated by Western blot,and the antigen-specificity of IgGsubclasses of Bactrian camel were further analyzed. The results showed that the IgG1,IgG2 and IgG3 of Bactrian camel are obtained successfully by the Protein A and Protein G affinity chromatography columns,the titers of the polyclonal antibodies of antiIgG1,IgG2,and IgG3 were all over 1∶ 10 000,and every polyclonal antibody had obvious cross-reactivity with IgG1,IgG2 and IgG3,but the polyclonal antibody specificity of anti-IgG1 was lower than those of anti-IgG2 and anti-IgG3. The results suggested that the immunogenicity of IgG1,IgG2 and IgG3 of Bactrian camel were all good and their specificities were all similar although their structures were obviously different.
引文
Alvarez-Rueda N,Behar G,FerréV,Pugnière M,Roquet F,Gastinel L,Jacquot C,Aubry J,Baty D,Barbet J,BirkléS.2007.Generation of llama single-domain antibodies against methotrexate,a prototypical hapten.Mol Immunol,44(7):1680-1690.
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De Genst E,Saerens D,Muyldermans S,Conrath K.2006a.Antibody repertoire development in camelids.Dev Comp Immunol,30(1-2):187-198.
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Decanniere K,Transue T R,Desmyter A,Maes D,Muyldermans S,Wyns L.2001.Degenerate interfaces in antigen-antibody complexes.J Mol Biol,313(3):473-478.
Desmyter A,Decanniere K,Muyldermans S,Wyns L.2001.Antigen specificity and high affinity binding provided by one single loop of a camel single-domain antibody.J Biol Chem,276(28):26285-26290.
Simone E A D,Saccodossi N,Ferrari A,Leoni J.2008.Development of ELISAs for the measurement of Ig M and Ig G subclasses in sera from llamas(Lama glama)and assessment of the humoral immune response against different antigens.Veterinary Immunology and Immunopathology,126(1):64-73.
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Maass D R,Sepulveda J,Pernthaner A,Shoemaker C B.2007.Alpaca(Lama pacos)as a convenient source of recombinant camelid heavy chain antibodies(VHHs).J Immunol Methods,324(1-2):13-25.
Muyldermans S,Atarhouch T,Saldanha J,Barbosa J A,Hamers.1994.Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains.Protein Eng,7(9):1129-1135.
Muyldermans S,Baral T N,Retamozzo V C,De Baetselier P,De Genst E,Kinne J,Leonhardt H,Magez S,Nguyen V K,Revets H,Rothbauer U,Stijlemans B,Tillib S,Wernery U,Wyns L,Hassanzadeh-Ghassabeh G,Saerens D.2009.Camelid immunoglobulins and nanobody technology.Vet Immunol Immunopathol,128(1-3):178-183.
Nguyen V K,Hamers R,Wyns L,Muyldermans S.1999.Loss of splice consensus signal is responsible for the removal of the entire C(H)1 domain of the functional camel IGG2A heavy-chain antibodies.Mol Immunol,36(8):515-524.
Nguyen V K,Hamers R,Wyns L,Muyldermans S.2000.Camel heavychain antibodies:diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire.EMBO J,19(5):921-930.
Nguyen V K,Muyldermans S,Hamers R.1998.The specific variable domain of camel heavy-chain antibodies is encoded in the germline.J Mol Biol,275(3):413-418.
van der Linden R,de Geus B,Stok W,Bos W,van Wassenaar D,Verrips T,Frenken L.2000.Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama.J Immunol Methods,240(1-2):185-195.
Verheesen P,Roussis A,de Haard H J,Groot A J,Stam J C,den Dunnen J T,Frants R R,Verkleij A J,Theo Verrips C,van der Maarel S M.2006.Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation.Biochim Biophys Acta,1764(8):1307-1319.
Vu K B,Ghahroudi M A,Wyns L,Muyldermans S.1997.Comparison of llama VH sequences from conventional and heavy chain antibodies.Mol Immunol,34(16-17):1121-1131.
Wine Y,Boutz D R,Lavinder J J,Miklos A E,Hughes R A,Hoi K H,Jung S T,Horton A P,Murrin E M,Ellington A D,Marcotte E M,Georgiou G.2013.Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response.Proc Natl Acad Sci USA,110(8):2993-2998.
Daley L P,Gagliardo L F,Duffy M S,Smith M C,Appleton JA.2005.Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids.Clin Diagn Lab Immunol,12(3):380-386.
De Genst E,Saerens D,Muyldermans S,Conrath K.2006a.Antibody repertoire development in camelids.Dev Comp Immunol,30(1-2):187-198.
De Genst E,Silence K,Decanniere K,Conrath K,Loris R,Kinne J,Muyldermans S,Wyns L.2006b.Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies.Proc Natl Acad Sci USA,103(12):4586-4591.
Decanniere K,Transue T R,Desmyter A,Maes D,Muyldermans S,Wyns L.2001.Degenerate interfaces in antigen-antibody complexes.J Mol Biol,313(3):473-478.
Desmyter A,Decanniere K,Muyldermans S,Wyns L.2001.Antigen specificity and high affinity binding provided by one single loop of a camel single-domain antibody.J Biol Chem,276(28):26285-26290.
Simone E A D,Saccodossi N,Ferrari A,Leoni J.2008.Development of ELISAs for the measurement of Ig M and Ig G subclasses in sera from llamas(Lama glama)and assessment of the humoral immune response against different antigens.Veterinary Immunology and Immunopathology,126(1):64-73.
Griffin L M,Snowden J R,Lawson A D,Wernery U,Kinne J,Baker T S.2014.Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species.J Immunol Methods,405(2):35-46.
Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa E B,Bendahman N,Hamers R.1993.Naturally occurring antibodies devoid of light chains.Nature,363(6428):446-448.
Lauwereys M,Arbabi Ghahroudi M,Desmyter A,Kinne J,H9lzer W,De Genst E,Wyns L,Muyldermans S.1998.Potent enzyme inhibitors derived from dromedary heavy-chain antibodies.EMBO J,17(13):3512-3520.
Lawson A D.2012.Antibody-enabled small-molecule drug discovery.Nat Rev Drug Discov,11(7):519-525.
Maass D R,Sepulveda J,Pernthaner A,Shoemaker C B.2007.Alpaca(Lama pacos)as a convenient source of recombinant camelid heavy chain antibodies(VHHs).J Immunol Methods,324(1-2):13-25.
Muyldermans S,Atarhouch T,Saldanha J,Barbosa J A,Hamers.1994.Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains.Protein Eng,7(9):1129-1135.
Muyldermans S,Baral T N,Retamozzo V C,De Baetselier P,De Genst E,Kinne J,Leonhardt H,Magez S,Nguyen V K,Revets H,Rothbauer U,Stijlemans B,Tillib S,Wernery U,Wyns L,Hassanzadeh-Ghassabeh G,Saerens D.2009.Camelid immunoglobulins and nanobody technology.Vet Immunol Immunopathol,128(1-3):178-183.
Nguyen V K,Hamers R,Wyns L,Muyldermans S.1999.Loss of splice consensus signal is responsible for the removal of the entire C(H)1 domain of the functional camel IGG2A heavy-chain antibodies.Mol Immunol,36(8):515-524.
Nguyen V K,Hamers R,Wyns L,Muyldermans S.2000.Camel heavychain antibodies:diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire.EMBO J,19(5):921-930.
Nguyen V K,Muyldermans S,Hamers R.1998.The specific variable domain of camel heavy-chain antibodies is encoded in the germline.J Mol Biol,275(3):413-418.
van der Linden R,de Geus B,Stok W,Bos W,van Wassenaar D,Verrips T,Frenken L.2000.Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama.J Immunol Methods,240(1-2):185-195.
Verheesen P,Roussis A,de Haard H J,Groot A J,Stam J C,den Dunnen J T,Frants R R,Verkleij A J,Theo Verrips C,van der Maarel S M.2006.Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation.Biochim Biophys Acta,1764(8):1307-1319.
Vu K B,Ghahroudi M A,Wyns L,Muyldermans S.1997.Comparison of llama VH sequences from conventional and heavy chain antibodies.Mol Immunol,34(16-17):1121-1131.
Wine Y,Boutz D R,Lavinder J J,Miklos A E,Hughes R A,Hoi K H,Jung S T,Horton A P,Murrin E M,Ellington A D,Marcotte E M,Georgiou G.2013.Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response.Proc Natl Acad Sci USA,110(8):2993-2998.