RESUMO
Objective:Mesenchymal stem cells( MSCs) have self-renewal capacity and potential to differentiate into the cells.It was reported that the expression of miR-30a changed in some immune diseases.But it remains unclear the effect of miR-30a on the im-munoregulatory functions of MSCs.Here we studied the impact of miR-30a on the phenotype,cell viability,apoptosis,cell cycle and im-munoregulatory functions of MSCs.Methods: The mixed enzyme methods were used for the isolation of human umbilical cord MSCs.Flow cytometry(FCM)was used to investigate the effect of overexpressed miR-30a on the phenotype of MSCs.CCK-8 was used to examine the cell viability of miR-30a-overexpressed MSCs.Annexin V/PI was used for the detection of apoptosis of MSCs.Q-PCR and Western blot were used to investigate the effect of miR-30a on the expression of Cyclin E2( CCNE2).CCNE2 was one putative target of miR-30a predicted by Targetscan database.The effects of miR-30a-overexpressed MSCs on the maturation of dendritic cells(DCs)were determined.Results:Overexpression of miR-30a blocked the cell cycle of MSCs in the G0/G1 phase by inhibiting the expression of CCNE2,but did not affect the phenotype, cell viability and apoptosis of MSCs.When co-cultured with DCs, although MSCs down-regulated the expression of CD40 and CD86 on DCs,overexpression of miR-30a more significantly enhanced the suppressive impact of MSCs on the maturation of DCs.Conclusion: miR-30a affects the cell cycle of MSCs and enhances its immunosuppressive effect on DCs.
RESUMO
FaeG is the key factor in the infection process of K88ad enterotoxigenic Escherichia coli(ETEC) fimbrial adhesin. In an attempt to determine the possibility of expressing recombinant FaeG with immunogenicity for a new safe and high-production vaccine in E. coli, we constructed the recombinant strain, BL21 (DE3+K88), which harbors an expression vector with a DNA fragment of faeG, without a signal peptide. Results of 15% SDS-polyacrylamide slab gel analysis showed that FaeG can be stably over-expressed in BL21 (DE3+K88) as inclusion bodies without FaeE. Immunoglobulin G (IgG) and M (IgM) responses in pregnant pigs, with boost injections of the purified recombinant FaeG, were detected 4 weeks later in the sera and colostrum. An in vitro villius-adhesion assay verified that the elicited antibodies in the sera of vaccinated pigs were capable of preventing the adhesion of K88ad ETEC to porcine intestinal receptors. The protective effect on the mortality rates of suckling piglets born to vaccinated mothers was also observed one week after oral challenge with the virulent ETEC strain, C83907 (K88ad, CT+, ST+). The results of this study proved that the adhesin of proteinaceous bacterial fimbriae or pili could be overexpressed in engineered E. coli strains, with protective immune responses to the pathogen.
Assuntos
Adesinas de Escherichia coli/imunologia , Fímbrias Bacterianas/imunologia , Imunoglobulina G/sangue , Proteínas Recombinantes/imunologia , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Animais , Animais Lactentes , Western Blotting , Ensaio de Imunoadsorção Enzimática , Vacinas contra Escherichia coli/genética , Vacinas contra Escherichia coli/imunologia , Vacinas contra Escherichia coli/isolamento & purificação , Feminino , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Imunização/métodos , Microscopia de Contraste de Fase , Gravidez , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SuínosRESUMO
Objective: Proto-oncogene Zbtb7A has been characterized as a molecular switch in the process of cancer initiation and development.Our goal is to obtain the POZ domain of the Zbtb7A protein and prepare its polyclonal antibodies.Methods: We optimized the coding sequence of the POZ domain according to the codon bias of E.coli and synthesized the sequence with two-step PCR,which was then introduced into the pET-26b(+) vector to express the protein.The recombinant protein was analyzed by 15% SDS-PAGE and the corresponding band was cut out from gel.The minced gel slice that contained the POZ domain protein was injected to immunize rabbits.The collected rabbit antiserum was purified using the saturated ammonium sulfate method in combination with protein G antibody purification,and the purified polyclonal antibodies was evaluated by Western blot.Results: The optimized sequence of the POZ domain was correctly obtained and successfully constructed into the pET-26b(+) vector.After induction,an expected protein band about 14 KD was detected on 15% SDS-PAGE,and highly purified polyclonal antibodies were obtained,which were specifically bound to human Zbtb7A.Conclusion: The obtained recombinant protein of the POZ domain and its polyclonal antibodies can be used for further studies of Zbtb7A.
