Effect of silencing MagT1 on the content of magnesium ion and apoptosis in rat cardiomyocytes / 重庆医学

Objective The changes of Mg2+ concentration and cell apoptosis were detected by using siRNA to silence MagT1 in rat cardio myocytes.Methods MagT1 siRNA sequences were designed and synthetized,then transfected into primary cultured rat myocardial cell for silencing MagT1.The expression of MagT1 mRNA and protein were detected by RT-PCR and Western blot.The changes of Mg2+ concentration in the cells were detected by fluorescence microscopy.Flow cytometry (FCM) was used to detect the cell apoptosis.Results Compared with negative siRNA group,MagT1 siRNA transfected rat cardiomyocytes after 48 h,MagT1 mRNA silence efficiency was 51.83 ％ (P＜0.05),the silence of MagT1 protein efficiency was 56.75 ％ (P＜0.05),intracellular Mg2+ concentration was reduced by 29.13％ (P＜0.05),the apoptosis rate was 31.18％ (P＜0.01);MagT1 siRNA transfected rat cardiomyocytes after 60 h,MagT1 mRNA silence efficiency was 86.91％ (P＜0.01),the silence of MagT1 protein efficiency was 83.85％ (P＜0.01),intracellular Mg2+ concentration was reduced by 41.32％ (P＜0.01),the apoptosis rate was 40.61％ (P＜0.01).Conclusion After the silencing of MagT1,the concentration of Mg2+ in the cells decreased significantly,the apoptotic rate increased significantly,cell life activities are greatly affected.

Preparation of monoclonal antibodies against recombinant Der f 7 allergen / 中国免疫学杂志

Objective:To clone and express the Der f 7 recombinant protein from Dermatophagoides farinae and prepare the Der f 7 monoclonal antibody.Methods: The Der f 7 recombinant protein was expressed in prokaryotic expression system of pET28a(+)-Der f 7.BALB /c mice were immunized with the recombinant protein.Myeloma cells and spleen cells were fused,and hybridoma cells were screened by ELISA.Hybridoma cells were injected into the mice abdominal cavity to obtain ascites.It was purified by protein A agarose medium ascites,and then to dentified the titer and purity of the antibody.The specificity of the antibody was identified by Western blot.Results: Three hybridoma cells which stably secret recombinant Der f 7 monoclonal antibody were obtained.The monoclonal antibody had high purity,the titer was higher than 1∶243 000.Western blot showed that Der f 7 recombinant protein could be recognized well.Conclusion: We successfully obtained Der f 7 monoclonal antibody,which can be used for the quantification and localization of Der f 7 allergen and the diagnosis and treatment of allergic diseases.

Structure and func tion comparation of dust mite allergen Der f 3,Der p 3 and Eur m 3 / 中国免疫学杂志

Objective:To compare structure and function of mite Der f 3,Der p 3 and Eur m 3.Methods: Obtained mite allergens amino acid sequences from the International Union of Immunological Societies nomenclature database .Then physiochemical characterization,sequence alignment,secondary structure,three dimensional (3D) structure and epitopes of three proteins were analyzed by bioinformatics methods.Results:According to results of alignment ,Der f 3,Der p 3 and Eur m 3 displayed 88.51%sequence iden-tity.Der f 3,Der p 3 and Eur m 3 all contained three active sites and two trypsin functional domains ,which showed high identity of amino acid residues.Active sites of three proteins ,which closing to each other in three dimensional (3D) structure,constituting the active center of the enzyme.Secondary and 3D structure of three proteins all contains α-helices,β-sheets and random coils.Epitopes analysis revealed that Der f 3,Der p 3 and Eur m 3 all have 5 main potential epitopes located in random coils.Epitope sequences of Der f 3,Der p 3 and Eur m 3 overlapping in three domains (peptides of 79-81aa,129-135aa and 172-174aa),but the residues in these three domains were not identical.Conclusion:These studies lay the foundation for biochemical and genetic analysis of these 3 allergens,and may contribute to vaccine development for allergen-specific immunotherapy.