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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.
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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.
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Objective: ①To detect the concentrations of interleukin 1?(IL 1?) and prostaglandin E 2 (PGE 2) in periapical exudates in the patients with acute or chronic periapical periodontitis; ② To examine their correlations with clinical and radiographic findings of the involved teeth.Methods: The volume of periapical exudates was quantified using the standard paper point sampling method; the concentration of IL 1? and PGE 2 was detected using enzyme linked immunosorbent assay(ELISA) or radioimmunoassay(RIA) in 40 cases with acute and in 40 with chronic periapical periodentitis. Results: ①The volume(?l) of the exudates in the cases with acute periapical periodontitis and chronic were 27.10?15.70 and 3.12?2.88( P
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Objective: To compare the concentration of IL-1? in the apical exudates of phoenix abscess of chronic periapical periodontitis and to examine the correlation of IL-I? concentration with clinical and radiographic findings of the involved teeth. Methods: 35 single-rooted teeth diagnosed as phoenix abscess and 35 as chronic periapical periodontitis were examined. The periapical signs and symptoms were recorded. Radiographs were taken and periapical radiolucent areas were calculated with the help of the AutoCAD software. The standard paper-point sampling method was used to collect and quantify the periapical exudates. IL-1? in the exudates was detected by enzyme-linked immunosorbent assay (ELISA). All statistical analyses were finished with SPSS 10.0 software. Results: The phoenix abscess group showed significantly lower concentration(5.65?2.76) ng/ml of IL-1? in the exudates and larger radiolucent areas(32.10?13.82) mm 2 on the X-ray films than the chronic periapical periodontitis group[(12.51?5.15) ng/ml and (6.51?3.56) mm 2 respectively] (P
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Macrofungi are rich in natural bioactive compounds so they have much medicinal value.Their immunomodulating and anticancer activity particularly attracts more attention.Based on the polysaccharides molecular structure and their relation to these activities,this paper discusses their mechanism of the immune responses and introduces the important instances of these polysaccharides along with their immunomodulating and/or anticancer activity isolated from putative medicinal mushrooms.
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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.