Ferritin is considered as an ideal delivery system due to its precise targeting, reversible self-assembly, high biocompatibility, and easy modification. this study aims to express, purify, and identify three fusion ferritin proteins, and explore their tumor targeting. Three fusion ferritin genes were synthesized and cloned into prokaryotic expression vectors, and the recombinant proteins were purified by affinity chromatography with nickel columns. The fusion ferritin proteins were identified by native polyacrylamide gel electrophoresis (native-PAGE), Western blotting, and circular dichroism. Fluorescein 5-isothiocyanate (FITC) was used to react with fusion ferritin, and confocal laser scanning microscopy was employed to evaluate the tumor targeting of fusion ferritin. The reaction system of sulfo-cyanine7 (Cy7-SE) with fusion ferritin was injected into the tail vein of melanoma mice for in vivo tumor imaging to explore the tumor targeting of fusion ferritin. The results showed that soluble fusion ferritin proteins of about 21 kDa were expressed under the induction by isopropylthio-β-d-galactoside (IPTG), and the recombinant proteins with high purity were obtained. Western blotting showed that the recombinant proteins could be recognized by the corresponding antibodies. The target proteins were identified as multimers with α helixes by native-PAGE and circular dichroism. In vitro and in vivo tumor uptake experiments demonstrated that fusion ferritin was taken up by tumor cells and tumor tissue. This study successfully expressed, purified, and identified fusion ferritin, and verified its tumor uptake in vitro and in vivo, which laid a foundation for the application of ferritin in biomedicine.
Animals ; Mice ; Recombinant Fusion Proteins/isolation & purification* ; Ferritins/metabolism* ; Escherichia coli/metabolism* ; Melanoma, Experimental/metabolism* ; Humans

OBJECTIVES: To analyze the clinical characteristics of fecal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid-positive in patients with coronavirus dasease 2019 (COVID-19) and to provide a scientific basis for the prevention and control of this disease. METHODS: The clinical data of 16 patients with fecal SARS-CoV-2 nucleic acid positive, who hospitalized in the North Branch of the First Hospital of Changsha (Changsha Public Health Rescue Center) from January to February 2020, were retrospectively analyzed. Their clinical manifestations, laboratory data and imaging data were summarized. RESULTS: Among the 16 patients, there were 9 males (56.25%) and 7 females (43.75%), the ratio of males to females was 1∶1.29. The age of onset was (43.3±14.6) years. There were 15 patients with contact history of Wuhan, 1 patient with contact history of local patient.Twelve patients were common type (75%), and 4 patients were severe type (25%). Clinical symptoms included fever in 14 patients (87.5%), cough in 12 patients (75%), shortness of breath in 5 patients (31.25%), pharyngalgia in 10 patients (62.5%), fatigue in 7 patients (43.75%), and diarrhea in 4 patients (25%). There were 14 patients (87.5%) with normal or decreased white blood cell count, 11 patients (68.75%) with decreased lymphocyte count, 15 patients (93.75%) with increased erythrocyte sedimentation rate, 13 patients (81.25%) with increased hypersensitivity C-reactive protein, 5 patients (31.25%) with increased procalcitonin, and 8 patients (50%) with increased serum ferritin in peripheral blood, and stool routine was basically normal. Compared with the common type, there was significant difference in the white blood cell and lymphocyte counts in the severe type (<0.01); the infection indicators, such as hypersensitivity C-reactive protein and serum ferritin, were significantly increased, with significant difference (all <0.01); but the procalcitonin and erythrocyte sedimentation rate was not significantly different (both >0.05). Chest CT mainly showed patchy shadows and interstitial changes. According to imaging examination, 4 patients (25%) showed unilateral pneumonia and 12 patients (75%) showed bilateral pneumonia. CONCLUSIONS The patients have the clinical symptoms of COVID-19, but gastrointestinal symptoms (such as diarrhea) are more common, and the changes of white blood cell count, lymphocyte count, hypersensitivity C-reactive protein, ferritin are more obvious in severe patients.The positivity of fecal nucleic acid suggests the possibility of digestive tract transmission of SARS-CoV-2, and fecal nucleic acid testing can be used as a routine testing method in clinical practice.
Adult ; Betacoronavirus ; isolation & purification ; C-Reactive Protein ; analysis ; China ; Coronavirus Infections ; diagnosis ; physiopathology ; Diarrhea ; virology ; Feces ; virology ; Female ; Ferritins ; analysis ; Humans ; Leukocyte Count ; Male ; Middle Aged ; Pandemics ; Pneumonia, Viral ; diagnosis ; physiopathology ; Retrospective Studies

The study was aimed to establish a new method of preparation of human placenta factor (PF) and to determine its physic-chemical properties, as well as effects on lymphocytes in vitro. PF was prepared by ultrafiltration. The contents and molecular weight of all constitutions were determined by Bradford method and SDS-PAGE, respectively. Cyclosporin A (CsA) was served as positive control, normal saline (NS) was used as negative control. PHA-stimulated lymphocyte proliferation and mixed lymphocyte reaction (MLR) were detected with MTT assay. The expression of CD69 on T cells was analyzed by flow cytometry. Cytotoxicity of natural killer (NK) cells against K562 tumor cells was examined with LDH release assay. The results indicated that PF was determined to be a group of low molecular weight polypeptides, consisting of two major components whose molecular weight were 9.187 and 4.794 kD respectively. The contents of PF were 5.7 - 6.9 mg/g fresh placenta. PF had similar suppressive effects on PHA-stimulated lymphocyte proliferation and MLR in vitro as compared with CsA (P > 0.05). Both PF and CsA could downregulate the expression of CD69 on T cells which had been stimulated by PMA plus ionomycin (PF vs CsA, P > 0.05). The cytotoxicity of NK cells against K562 cells in PF group was slightly higher or equivalent as compared with that in NS group (P > 0.05), but the cytotoxicity in CsA group was much lower than that in NS group (P < 0.05). It is concluded that a new method of preparation of PF has been established. This study first demonstrates that PF has strong immunosuppressive effects on T cell in vitro, and suppresses T cell proliferation and activation induced by mitogen and alloantigen. This study indicats that PF has no any inhibitory effects, but even enhances the cytotoxicity of NK cells against K562 tumor cells. These results suggest that PF may have suppressive effects on graft-versus-host disease (GVHD) without diminishing graft-versus-tumor (GVT) effects. Therefore, PF may probably be an ideal and promising agent against GVHD.
Cyclosporine ; pharmacology ; Ferritins ; immunology ; isolation & purification ; Graft vs Host Disease ; metabolism ; prevention & control ; Humans ; Immunosuppressive Agents ; immunology ; K562 Cells ; Killer Cells, Natural ; immunology ; Lymphocytes ; immunology ; T-Lymphocytes ; immunology

To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell, 8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst (P<0.05). PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics (P>0.05). It was concluded that PLF at the concentration of 10-100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantation blastocysts.
Coculture Techniques ; Decidua/*cytology ; Embryo, Mammalian/*cytology ; Embryo, Mammalian/drug effects ; Embryo, Mammalian/embryology ; Embryonic Development/*drug effects ; Ferritins/isolation & purification ; Ferritins/*pharmacology ; Placenta/*chemistry ; Tissue Culture Techniques