鸡恒定链协助抗原肽结合MHCⅡ类分子并进入细胞内吞体
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摘要
为探明鸡恒定链(invariant chain,Ii)的胞质区/跨膜区[Ii(Cyt/Tra)]作为载体是否可携带抗原肽在细胞内结合MHCⅡ类分子和进入转运途径的细胞器(内吞体),构建4个基因目的片段[Ii、Ii(Cyt/Tra)、F2(新城疫病毒F蛋白片段)和Ii(Cyt/Tra)/F2],分别将它们插入原核表达载体pET-32a和真核表达载体pmCherry-C1/N1中,构建8个重组质粒,再转入工程菌(E.coli)和人肾细胞系(293T),并应用拉下法(pull-down)检测目的蛋白与MHCⅡβ的结合,应用激光共聚焦确定它们在真核细胞与MHCⅡβ的共定位以及在内吞体的定位。结果表明,构建的重组质粒均能相应地原核或真核表达相应目的蛋白;Ii、Ii(Cyt/Tra)和Ii(Cyt/Tra)/F2不仅能够与MHCⅡβ结合,还能进入细胞内吞体;但是F2既不能与MHCⅡβ结合,也不能进入内吞体。Ii活性片段Cyt/Tra不仅本身具有结合MHCⅡβ的作用,而且还可以携带抗原肽与之结合并一起转入细胞内吞体而进入抗原递呈途径。该结果为进一步研究Ii载体转运抗原提供了理论依据。
This experiment was conducted to clear if chicken invariant chain(Ii)cytoplasmic/transmembrane domains—Ii(Cyt/Tra),as immune carrier—is able to carry antigen peptide to associate with MHC classⅡ molecules and enter the endosomes in cell transport pathway.In this study,4 gene segments,such as Ii,Ii(Cyt/Tra),F2(Newcastle disease virus F protein fragment)and Ii(Cyt/Tra)/F2,were amplified and constructed by PCR.They were inserted into prokaryotic expression plasmid pET-32 aand eukaryotic expression plasmid pmCherry-C1/N1 respectively,and8 recombinant plasmids were constructed.Then they were transferred into the engineering bacteria(E.coli)and human renal cell(293 T)respectively.The binding of the target protein to the MHCⅡβwas detected by pull-down assay,and the co-localization in the eukaryotic cells with the MHCⅡβand the localization in the endosomes were determined by laser confocal.The results showed that all the recombinant target protein molecules were expressed in prokaryotic or eukaryotes;Ii,Ii(Cyt/Tra)and Ii(Cyt/Tra)/F2 were able to bind to MHCⅡβas well as to enter endosomes except F2.To sum up,the active domains,Ii(Cyt/Tra),not only itself had the effect of associating MHCⅡβ,but also could bring antigenic peptides to bind MHCⅡβand transport the endosome together,and finally enter the antigen presentation pathway.This provides a theoretical basis for further study of Ii as immune carrier.
This experiment was conducted to clear if chicken invariant chain(Ii)cytoplasmic/transmembrane domains—Ii(Cyt/Tra),as immune carrier—is able to carry antigen peptide to associate with MHC classⅡ molecules and enter the endosomes in cell transport pathway.In this study,4 gene segments,such as Ii,Ii(Cyt/Tra),F2(Newcastle disease virus F protein fragment)and Ii(Cyt/Tra)/F2,were amplified and constructed by PCR.They were inserted into prokaryotic expression plasmid pET-32 aand eukaryotic expression plasmid pmCherry-C1/N1 respectively,and8 recombinant plasmids were constructed.Then they were transferred into the engineering bacteria(E.coli)and human renal cell(293 T)respectively.The binding of the target protein to the MHCⅡβwas detected by pull-down assay,and the co-localization in the eukaryotic cells with the MHCⅡβand the localization in the endosomes were determined by laser confocal.The results showed that all the recombinant target protein molecules were expressed in prokaryotic or eukaryotes;Ii,Ii(Cyt/Tra)and Ii(Cyt/Tra)/F2 were able to bind to MHCⅡβas well as to enter endosomes except F2.To sum up,the active domains,Ii(Cyt/Tra),not only itself had the effect of associating MHCⅡβ,but also could bring antigenic peptides to bind MHCⅡβand transport the endosome together,and finally enter the antigen presentation pathway.This provides a theoretical basis for further study of Ii as immune carrier.
引文
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[14]王琛,刘雪兰,陈芳芳,等.鸡恒定链功能片段跨物种增强小鼠对新城疫F2抗原免疫作用的研究[J].西北农林科技大学学报:自然科学版,2014,42(5):33-38.WANG C,LIU X L,CHEN F F,et al.Cross species enhancement of mice antigen immune function against NDV-F2 by invariant chain functional fragments of chicken[J].Journal of Northwest A&F U-niversity:Natural Science Edition,2014,42(5):33-38.(in Chinese)
[15]CHEN F F,PAN L,ZHANG J G,et al.Allele-dependent association of chicken MHC class I molecules with the invariant chain[J].Vet Immunol Immunopathol,2014,160(3-4):273-280.
[16]CHEN F F,WU C,PAN L,et al.Cross-species association of quail invariant chain with chicken and mouse MHCⅡmolecules[J].Dev Comp Immunol,2013,40(1):20-27.
[17]CHEN F F,LIN H B,LI J C,et al.Grass carp(Ctenopharyngodon idellus)invariant chain of the MHC class II chaperone protein associates with the class I molecule[J].Fish Shellfish Immunol,2017,63:1-8.
[18]ROMAGNOLI P,GERMAIN R N.The CLIP region of invariant chain plays a critical role in regulating major histocompatibility complex class II folding,transport,and peptide occupancy[J].J Exp Med,1994,180(3):1107-1113.
[19]CHEN F,PAN L,CHAO W,et al.Character of chicken polymorphic major histocompatibility complex class II alleles of 3Chinese local breeds[J].Poultry Sci,2012,91(5):1097-1104.
[20]BIDDISON W E,RAO P E,TALLE M A,et al.Possible involvement of the T4molecule in T cell recognition of class II HLA antigens:evidence from studies of proliferative responses to SB antigens[J].JImmunol,1983,131(1):152-157.
[21]O'SULLIVAN D M,NOONAN D,QUARANTAV.Four Ia invariant chain forms derive from a single gene by alternate splicing and alternate initiation of transcription/translation[J].J Exp Med,1987,166(2):444-460.
[22]许发芝,吴胜国,刘雪兰,等.鸡恒定链分子跨膜区2个氨基酸残基在形成MHCⅡ-Ii复合物中的作用[J].畜牧兽医学报,2011,42(5):721-728.XU F Z,WU S G,LIU X L,et al.The function of two amino acid residues located in chicken invariant chain transmembrane domain in assembly of MHCⅡ-Ii complex[J].Acta Veterinaria et Zootechnica Sinica,2011,42(5):721-728.(in Chinese)
[23]罗兰芳,余为一,陈芳芳.鸡Ii与B-L基因表达产物的细胞定位与结合活性特征[J].中国免疫学杂志,2015,31(7):879-883,889.LUO L F,YU W Y,CHEN F F.Characters of location in cells and binding activity of chicken Ii and B-Lgene ex-pressed products[J].Chinese Journal of Immunology,2015,31(7):879-883,889.(in Chinese)
[24]LANDSVERK O J,BAKKE O,GREGERS T F.MHC II and the endocytic pathway:regulation by invariant chain[J].Scand J Immunol,2009,70(3):184-193.
[25]PIETERS J,HORSTMANN H,BAKKE O,et al.Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain[J].J Cell Biol,1991,115(5):1213-1223.
[26]KOLESNIKOVA L,BERGHFER B,BAMBERGS,et al.Multivesicular bodies as a platform for formation of the marburg virus envelope[J].J Viol,2004,78(22):12277-12287.
[2]CHEN F,PAN L,DAI Y,et al.Characteristics of expression of goose invariant chain gene and comparison of its structure among different species[J].Poultry Sci,2011,90(8):1664-1670.
[3]ZHONG D L,YU W Y,BAO M,et al.Molecular cloning and mRNA expression of duck invariant chain[J].Vet Immunol Immunopathol,2006,110(3-4):293-302.
[4]STOCKINGER B,PESSARA U,LIN R H,et al.Arole of la-associated invariant chains in antigen processing and pressentation[J].Cell,1989,56(4):683-689.
[5]BIJLMAKERS M J,BENAROCH P,PLOEGH HL.Mapping functional regions in the lumenal domain of the class II-associated invariant chain[J].J Exp Med,1994,180(2):623-629.
[6]GENVE L,CHEMALI M,DESJARDINS M,et al.Human invariant chain isoform p35restores thymic selection and antigen presentation in CD74-deficient mice[J].Immunol Cell Biol,2012,90(9):896-902.
[7]BREMNES B,MADSEN T,GEDDE-DAHL M,et al.An LI and ML motif in the cytoplasmic tail of the MHC-associated invariant chain mediate rapid internalization[J].J Cell Sci,1994,107(Pt 7):2021-2032.
[8]STARODUBOVA E S,ISAGULIANTS M G,KUZ-MENKO Y V,et al.Fusion to the lysosome targeting signal of the invariant chain alters the processing and enhances the immunogenicity of HIV-1reverse transcriptase[J].Acta Nat,2014,6(1):61-68.
[9]JENSEN B A H,STEFFENSEN M A,NIELSEN KN,et al.Co-expression of tumor antigen and interleukin-2from an adenoviral vector augments the efficiency of therapeutic tumor vaccination[J].Mol Ther,2014,22(12):2107-2117.
[10]MIKKELSEN M,HOLST P J,BUKH J,et al.Enhanced and sustained CD8+T cell responses with an adenoviral vector-based hepatitis C virus vaccine encoding NS3linked to the MHC class II chaperone protein invariant chain[J].J Immunol,2011,186(4):2355-2364.
[11]XIONG R,DAI W,YU W Y,et al.Influence of the Series Multiepitopes of DNV-HN and IBDV-VP2on an immune enhancement mediated by invariant chain segments as carrier[J].J Anim Vet Adv,2014,13(4):236-240.
[12]CHEN F F,MENG F T,PAN L,et al.Boosting immune response with the invariant chain segments via association with non-peptide binding region of major histocompatibility complex class II molecules[J].BMC Immunol,2012,13:55.
[13]TOYODA T,GOTOH B,SAKAGUCHI T,et al.Identification of amino acids relevant to three antigenic determinants on the fusion protein of Newcastle disease virus that are involved in fusion inhibition and neutralization[J].J Virol,1988,62(11):4427-4430.
[14]王琛,刘雪兰,陈芳芳,等.鸡恒定链功能片段跨物种增强小鼠对新城疫F2抗原免疫作用的研究[J].西北农林科技大学学报:自然科学版,2014,42(5):33-38.WANG C,LIU X L,CHEN F F,et al.Cross species enhancement of mice antigen immune function against NDV-F2 by invariant chain functional fragments of chicken[J].Journal of Northwest A&F U-niversity:Natural Science Edition,2014,42(5):33-38.(in Chinese)
[15]CHEN F F,PAN L,ZHANG J G,et al.Allele-dependent association of chicken MHC class I molecules with the invariant chain[J].Vet Immunol Immunopathol,2014,160(3-4):273-280.
[16]CHEN F F,WU C,PAN L,et al.Cross-species association of quail invariant chain with chicken and mouse MHCⅡmolecules[J].Dev Comp Immunol,2013,40(1):20-27.
[17]CHEN F F,LIN H B,LI J C,et al.Grass carp(Ctenopharyngodon idellus)invariant chain of the MHC class II chaperone protein associates with the class I molecule[J].Fish Shellfish Immunol,2017,63:1-8.
[18]ROMAGNOLI P,GERMAIN R N.The CLIP region of invariant chain plays a critical role in regulating major histocompatibility complex class II folding,transport,and peptide occupancy[J].J Exp Med,1994,180(3):1107-1113.
[19]CHEN F,PAN L,CHAO W,et al.Character of chicken polymorphic major histocompatibility complex class II alleles of 3Chinese local breeds[J].Poultry Sci,2012,91(5):1097-1104.
[20]BIDDISON W E,RAO P E,TALLE M A,et al.Possible involvement of the T4molecule in T cell recognition of class II HLA antigens:evidence from studies of proliferative responses to SB antigens[J].JImmunol,1983,131(1):152-157.
[21]O'SULLIVAN D M,NOONAN D,QUARANTAV.Four Ia invariant chain forms derive from a single gene by alternate splicing and alternate initiation of transcription/translation[J].J Exp Med,1987,166(2):444-460.
[22]许发芝,吴胜国,刘雪兰,等.鸡恒定链分子跨膜区2个氨基酸残基在形成MHCⅡ-Ii复合物中的作用[J].畜牧兽医学报,2011,42(5):721-728.XU F Z,WU S G,LIU X L,et al.The function of two amino acid residues located in chicken invariant chain transmembrane domain in assembly of MHCⅡ-Ii complex[J].Acta Veterinaria et Zootechnica Sinica,2011,42(5):721-728.(in Chinese)
[23]罗兰芳,余为一,陈芳芳.鸡Ii与B-L基因表达产物的细胞定位与结合活性特征[J].中国免疫学杂志,2015,31(7):879-883,889.LUO L F,YU W Y,CHEN F F.Characters of location in cells and binding activity of chicken Ii and B-Lgene ex-pressed products[J].Chinese Journal of Immunology,2015,31(7):879-883,889.(in Chinese)
[24]LANDSVERK O J,BAKKE O,GREGERS T F.MHC II and the endocytic pathway:regulation by invariant chain[J].Scand J Immunol,2009,70(3):184-193.
[25]PIETERS J,HORSTMANN H,BAKKE O,et al.Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain[J].J Cell Biol,1991,115(5):1213-1223.
[26]KOLESNIKOVA L,BERGHFER B,BAMBERGS,et al.Multivesicular bodies as a platform for formation of the marburg virus envelope[J].J Viol,2004,78(22):12277-12287.