ABSTRACT
Objective:To analyze the subcellular localization of family of serine hydrolases 1 (FSH1) protein in Microsporum canis. Methods:The FSH1 and enhanced green fluorescent protein (EGFP) genes were amplified by PCR using the previously constructed plasmid containing the FSH1 gene and the recombinant plasmid pCAMBIA-LRP-EGFP as the template; the vector DNA was obtained by double-enzyme digestion of the recombinant plasmid pCAMBIA-LRP-EGFP with SnaBI/KpnI. Then, the EGFP expression plasmid and Ptrcp-FSH1-EGHP-Ttrcp fusion plasmid were constructed by inserting the amplified EGFP gene and EGFP-FSH1 gene into the vector DNA respectively, and identified by PCR and sequencing. The two recombinant plasmids were transformed into Microsporum canis by an Agrobacterium tumefaciens-mediated method, and the gene EGFP and fusion gene FSH1-EGFP were expressed integratedly in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc. The cellular localization of the fusion protein was observed by laser scanning confocal microscopy. Results:The Agrobacterium tumefaciens-mediated transformation system and EGFP expression vector in Microsporum canis were successfully constructed; the fusion gene FSH1-EGFP was expressed integratedly in Microsporum canis. Laser confocal microscopy showed that fluorescence signals of the FSH1-EGFP fusion protein were concentrated in the cytoplasm and nuclei of Microsporum canis, with a granular or cluster-like appearance. Conclusion:The FSH1-EGFP fusion protein was successfully localized in the cytoplasm and nuclei of Microsporum canis, providing a basis for further clarifying the function and pathogenic mechanisms of the FSH1 gene in Microsporum canis.
ABSTRACT
Objective To determine the location of PQ-LRP protein in Microsporum canis using the enhanced green fluorescent protein(EGFP)as a marker.Methods The total RNA was extracted from Microsporum canis,and reversely transcribed into cDNA.The PQ-LRP gene was amplified by PCR using the above cDNA as the template.The fusion gene of PQ-LRP gene and EGFP gene was linked to the plasmid pCAMBIA 1300.Microsporum canis was subjected to Agrobacterium tumefaciens-mediated transformation,in order to achieve the integrated expression of the fusion gene LRP-EGFP in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc.Laser-scanning confocal microscopy was conducted to determine the cellular localization of the fusion protein.Results The expression vector pCAMBIA-LRP-EGFP was successfully constructed,and the fusion gene LRP-EGFP was expressed integratedly in Microsporum canis.Laser-scanning confocal microscopy showed that fluorescence signals of LRP-EGFP were concentrated on the cell membrane of Microsporum canis,giving a granular or cluster-like appearance.Conclusion The infusion gene LRP-EGFP can be successfully expressed in Microsporum canis,and PQ-LRP protein is located on the cell membrane of Microsporum canis.
ABSTRACT
BACKGROUND:Bone marrow is the main source of mesenchymal stem cells(MSCs)at present,but its application has been limited,because of some reasons such as inconvenience of isolation,and the quantity of cells decreases with human increased age.Umbilical cord as a new source of MSCs has been widespread concerned recently.OBJECTIVE:To explore the approach of isolating and culturing MSCs from Wharton's jelly of human umbilical cord,and the methods of identifying the surface antigens and the differentiation potential.METHODS:MSCs were isolated and amplified via tissue-cultivation,and cultured by FasGrow medium.Morphology of MSCs from Wharton's jelly of human umbilical cord was observed under the optical microscope.Its immunophenotypes were detected using immunohistochemistry.The differentiation of MSCs into the osteoblasts was determined utilizing Gomori calcium-cobalt alkaline phosphatase staining,von Kossa calcium node staining,and tetracyclinefluorescence labeling.The differentiation of MSCs into the adipocytes was detected using oil red O staining.RESULTS AND CONCLUSION:MSCs were easily obtained from Wharton's jelly of human umbilical cord via the proposed approach.The primary cells grew up to 70%-80% confluence after 12-16 days of culture,and meanwhile the undifferentiated state was maintained and proliferation was stabilized after passage.The cell cycle of double increase was about 2 days,and proliferation in vitro reached twenty generation above.Surface antigen analysis showed that CD44,CD105,CD133,MHC-I were positively expressed,while CD34,CD45 were negatively expressed.Experiments of differentiation in vitro indicated that the obtained cells were capable of differentiating into fat,osteoblast and nerve-like cells.