ABSTRACT
Objective To investigate the therapeutic effect of KGF-2 mutants on radiation-induced intestinal mucosal injury.Methods Specific pathogen free (SPF) female C57BL/6J mice were randomly divided into 4 groups, which were treated with/without KGF2 mutant and/or bone marrow cell transplantation.All the 4 groups were radiated with one-time whole bodyγ-ray exposure of 12 Gy.Also one untreated group was included as control.The therapeutic effect of recombi-nant human KGF-2 mutants on radiation-induced intestinal mucosal injury was analyzed by the survival rate at the 30th day, the pathological change and the apoptosis as well as autophagy status of small intestine at 72 hours after exposure.Results After irradiation, all the mice in the untreated group died within 9 days while the mice treated with KGF-2 mutant combined with bone marrow cell transplantation showed a 70%survival rate at the 30th day.We also found that intestinal mucosa of the mice in this group had a more intact structure, a higher level of autophagy and a lower level of apoptosis via HE staining and immunohistochemistry.Conclusion KGF-2 mutant has a significant therapeutic effect on radiation-induced intestinal mucosal injury.
ABSTRACT
The cDNA of Insulin-like growth factor binding protein 3 was subcloned into a eukaryotic secretory expression vector pSectagA to construct pSectag-IGFBP3. Human renal cell carcinoma (RCC) 786-0 cells were transfected with pSectag-IGFBP3 using lipofectamine 2000. After 48 h, the secretory IGFBP-3 was tested and identified by western blotting. Meanwhile, Annexin V-EGFP stain was used to analyze the apoptosis of 786-0 cells induced by IGFBP-3. Secretory IGFBP-3 protein could express successfully in the 786-0 cells and the expressed IGFBP-3 directly displayed an apoptotic effect on the host cells. This work provides a basis for further study on the apoptosis-inducing mechanism of IGFBP-3 and the development of a new anti-tumor drug.
Subject(s)
Humans , Apoptosis , Base Sequence , Carcinoma, Renal Cell , Metabolism , Pathology , Insulin-Like Growth Factor Binding Protein 3 , Genetics , Bodily Secretions , Kidney Neoplasms , Metabolism , Pathology , Molecular Sequence Data , Recombinant Fusion Proteins , Genetics , Physiology , Transfection , Tumor Cells, CulturedABSTRACT
Both interleukin-1 and IgE are important in the pathogenic mechanism of the allergy asthma. cDNA of interleukin-1receptor antagonist (IL-1ra) and IgE were cloned and a prokaryotic expression vector IL-1ra-Fcepsilon/pBV220 was constructed. The vector was transformed into Escherichia coli BL21(DE3). The fusion protein was expressed successfully in the form of inclusion body. The recombination protein of IL-1ra-Fcepsilon was highly purified by chromatography of gel filtration and ion exchange, which was identifited by Western blotting. The cell assay showed that the activity of IL-1ra-Fcepsilon was as high as IL-1ra in vitro after refolding. The pharmacokenetic profile of IL-1ra-Fcepsilon and L-1ra was analyzed, and the half time of IL-1ra-Fcepsilon is 4.78 times than that of IL-1ra.
Subject(s)
Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Gene Fusion , Immunoglobulin E , Genetics , Metabolism , Immunoglobulin Fc Fragments , Genetics , Metabolism , Interleukin 1 Receptor Antagonist Protein , Genetics , Metabolism , Recombinant Fusion Proteins , Genetics , MetabolismABSTRACT
Objective To study on the immunogenicity of SAK and its mutant using PAb.Methods 4 New Zealand white rabbits subcutaneously injected with corresponding antigens were grouped into group SAK involving 2 rabbits and group SAK2 involving 2 rabbits.The antiserums were collected 1 week after third injection.The rabbit IgG fraction was precipitated with saturated(NH4)2SO4 and purified by DEAE-Sepharose column chromatography,and then the antibody titer was determined by ELISA.The immunoreactivity of antigen to polyclonal antibody against SAK and SAK2 was tested using ELISA.Results The immunoreactivity of SAK2 to polyclonal antibody against SAK was sharply reduced to a very low level determined by ELISA,which indicated that some epitopes of SAK had been deleted.Conclusions The immunoreactivity of SAK to polyclonal antibody against SAK2 did not get a dramatically change compared with SAK2,which indicated that the reconstruction of epitope did not create new epitope.
ABSTRACT
Objective:To map dominant antigenic determinants on SAK by gene-targeted fragmemt library . Methods:①The PcAbs specificly against SAK were produced by immunning BALB/C mice. Pu rified the antisera through a SAK-Sepharose 4B affinity chromatography column. The purified PcAbs were biotinylated for next step. ② After constructing SAK r andom epitope library we sequenced 12 isolated clones randomly to ensure its int egrity ,capability and randomness.③The library was screening by situ-clone hy bridization.④Constructed mSAK ,the A1 region deleted mutant of SAK ,and used Western-blot assay to identified its immunoreactivity. Results:①Got a dominant epitope at amino acid 71-89,called A1 region; ②Western-bl ot assay suggested that mSAK, a mutant SAK without A1 region., didn't combine to the anti-SAK PcAbs.Conclusion:A dominant epitope of SAK was mapped successfully with a simple,effective method . [