Research of functional segments of chicken B-FA molecule binding with Ii chain / 中国免疫学杂志

Objective: To research the functional segments of B-FA molecule binding invariant chain and their characters. Methods:The DNA segments (α1α2, sα1α2 and α3TC ) of B-FA genes were respectively cloned and inserted into prokaryotic or eukaryotic expression plasmids,then they were singly or co-transfected with Ii gene into the engineering bacteria E. coli (BL-21)or 293T cells. After induction of expression,affinity chromatography and SDS-PAGE identification,the binding between B-FA segments and Ii molecule and co-localization in cells were observed with Pull-down and Western blot. Results:First three recombinant prokaryotic expression plasmids and four recombinant eukaryotic expression plasmids were constructed. The single molecules expressed by B-FA segments were observed after an affinity chromatography. Secondly the complexes of Ii/B-FA-α1α2 and Ii/B-FA-sα1α2 were detected by a Pull-down from the co-transfected corresponding prokaryotic expression plasmids,but no complex of Ii andα3TC,also in the western blot it was detected that B-FA-α1α2 or B-FA-sα1α2 as functional segment could bind Ii to form complex. Finally in eukaryotic expression 293T cells B-FA-sα1α2 kept localization, the same as B-FA. Conclusion: Chicken B-FA-α1α2 is function segment to bind with Ii molecule and keeps the location characters same as B-FA. The results of this research first time provide experimental evidence about B-FA functional region binding segment to Ii molecule.

Characters of location in cells and binding activity of chicken Ii and B-L gene ex-pressed products / 中国免疫学杂志

Objective:To study characters of the location in cells and binding activity of chicken Ii and B-L gene expressed products.Methods:The cloned gene segments of chicken Ii,B-LA and B-LB were respectively inserted into prokaryotic or eukaryotic expression plasmids,and then these recombinant plasmids were respectively alone transfected or cotransfected into engineering bacteria, Rosetta(DE3) or 293T cells.All of the recombinant bacteria were induced to express and their products then were renatured.The singly expressed products were detected to their immunogenicity with Western blot, and the co-expressed products were tested their binding with pull-down method and ( SDS-) PAGE.Results:First six of prokaryotic and eukaryotic recombinant expression plasmids were con-structed.The eukaryotic expressed products of Ii, B-LA and B-LB genes located in cellular plasma membrane.The single protein molecules were achieved from prokaryotic expressed products, which were renatured and purified with a Ni-column respectively.Secondly the prokaryotic expression Ii,B-LA and B-LB molecules could respectively induce mouse to product specific anti-bodies, which could recognize the corresponding products in eukaryotic expression.This indicated that they retained their antigenicity.Finally with pull-down from the products in co-expression in engineering bacteria and renaturation the Ii/B-LA or Ii/B-LB complexes were purified and the Ii,B-LA or B-LB monomers were dissociated from these complexes after a SDS treatment.Conclusion:The prokaryotic or eukaryotic expressed products of chicken Ii and B-L genes could retain their antigenicity,and chicken Ii could bind B-L molecules after prokaryotic coexpression and renaturation.These results provide a useful method to study the relation between Ii and MHC molecules.

Hallazgo de patrones para péptidos-vacuna con capacidad de acople universal en moléculas de HLA-II / Identification of patterns for peptide-vaccines in universal capacity coupling HLA-II molecules / Identificagáo de padroes para peptídeos-vacina com capacidade de acoplamento universal em moléculas HLA-II

Partiendo del alineamiento múltiple de secuencias proteicas humanas obtenidas de las bases de datos del National Center of Biotechnology Information (NCBI) y su posterior análisis espacial tridimensional, se estableció la existencia de un patrón de acople universal para péptidos presentados por las moléculas de histocompatibilidad HLA-II (DR, DP y DQ), siendo una base para el diseño de vacunas proteicas. Estos patrones espaciales fueron claramente exhibidos por los residuos altamente conservados de los tres tipos de moléculas de HLA-II. La aplicación de este nuevo hallazgo permitió diseñar péptidos con mejores valores de acople péptido-HLA-II, que los generados por el péptido de acople universal conocido como CLIP (class Il-associated invariant chain peptide).

Starting from the multiple alignment of human protein sequences obtained from the NCBI database (National Center of Biotechnology Information) and subsequent three-dimensional spatial analysis, the existence of a pattern of universal coupling to peptides presented by MHC molecules HLA-II (DR, DP and DQ) was established, being a basis for the design of protein vaccines. These spatial patterns were clearly exhibited by highly conserved residues of the three kinds of HLA-II molecules. The application of this new finding made it possible to design peptides with better Peptide -HLA-II coupling values than those generated by the universal coupling peptide called CLIP (class II-associated invariant chain peptide).

FA partir do alinhamento múltiplo de sequéncias de proteínas humanas obtidas a partir das bases de dados do NCBI (National Center of Biotechnology Information) e análise espacial tridimensional subsequente, estabeleceu-se a existéncia de um padráo de acoplamento universal para peptídeos apresentados pelas moléculas de histocompatibilidade HLA-II (DR, DP e DQ), sendo uma base para o desenho de vacinas proteicas. Estes padroes espaciais foram claramente exibidos pelos residuos altamente conservados dos trés tipos de moléculas de HLA-II. A aplicagáo deste novo achado permitiu desenhar peptídeos com melhores valores de acoplamento peptídeo-HLA-II, do que aqueles gerados pelo peptídeo de acoplamento universal conhecido como CLIP (classe II-peptídeo associado a cadeia invariante).

The intracellular localization and association of chicken major histocompatibility complex classⅠsubunits with invariant chain / 中国免疫学杂志

Objective:To investigate the intracellular localization and association of chicken major histocompatibility complex class Ⅰsubunits with invariant chain.Methods:At first,the sequences of MHCⅠ ? and ?2m subunits were amplified with RT-PCR.Secondly,we constructed the eukaryotic expression plasmids of MHCⅠ ? and MHCⅠ?2m that was fused with red or enhanced green fluorescent protein,respectively.The recombinant plasmids of MHC Ⅰsubunits and pEGFP-C1-Ii that could express enhanced green fluorescent protein were transiently transfected into COS-7 cells with Lipofectamine 2000.Immunofluorescence microscopy was carried out to detect the expression and intracellular localization of Ii and MHC Ⅰsubunits,and immunoprecipitation was used for analyzing their association.Results:The colocalization was found in endocytic compartments when GFP-Ii,MHCⅠ?-RFP and MHCⅠ?2m-RFP were co-expressed in COS-7 cells.Immunoprecipitation of Ii showed that GFP-Ii co-isolated with MHCⅠ?-GFP and MHCⅠ?2m-GFP subunits when COS-7 cells were transiently transfected with pEGFP-C1-Ii,pEGFP-N1-MHCⅠ? and pEGFP-N1-MHCⅠ?2m.Conclusion:Chicken Ii can not effectively associate with single ? or ?2m subunits from chicken MHC Ⅰmolecules,but Ii and the integrated MHC Ⅰmolecule can form the complexes that are colocalized in endomembrane system of COS-7 cells.