Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This co...Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface.Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 SDF-1KDEL,pcDNA3.1 SDF-154KDEL,pEGFPSDF-1KDEL and pEGFPSDF-154KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate,they were transferred into COS-7 cells with liposome . The exogenous expressions were observed, fusion protein SDF-1His and SDF-154His were confirmed by Western blot,and the SDF-1EGFP and SDF-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1 KDELHis and SDF-1 54 KDELHis expressed in COS-7 cells.Subcelluar localization analysis showed that SDF-1KDELEGFP and SDF-154KDELEGFP were located mainly in endoplasmic reticulum.Conclusion:Four expression vectors pcDNA3.1 SDF-1KDEL, pcDNA3.1SDF-154KDEL, pEGFPSDF-1KDEL and pEGFPSDF-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .展开更多
Objective:To prepare folate liposome complexes of recombinant plasmid pEGFP/SDF-1/KDEL wich contained intrakine SDF-1/KDEL gene and to observe its targeting for breast cancer cells.Methods:Reverse phase evaporation me...Objective:To prepare folate liposome complexes of recombinant plasmid pEGFP/SDF-1/KDEL wich contained intrakine SDF-1/KDEL gene and to observe its targeting for breast cancer cells.Methods:Reverse phase evaporation method was employed for preparing folate-liposome complexeses of pEGFP/SDF-1/KDEL,so that preparation condition was optimized;and fluorometric method was taken for testing encapsulation efficiency.Particle diameter of liposome was tested by transmission electron microscopy.Complexeses encapsulated with 0.05-0.25 mg/ml calcein were got and incubated with breast cancer cell line MDA-MB-231 cells for 2 to 12 h,then dissolved them with dimethyl sulfoxide,then detected absorbance with microplate reader.Results:The optimized encapsulation condition are as follows:molecular ratio of lecithin and cholesterol was 3:1,rotary speed was 150 r/min under temperature of 43℃,and encapsulation efficiency reached 81%in this experiment.Average liposome particle diameter was 210 nm;8 h after incubation, the intake of MDA-MB-231 cells to 0.25 mg/ml compounds achieved saturation. Conclusion:The pEGFP/SDF-1/KDEL folic acid liposome complexes prepared has a high encapsulating rate,liposome particle diameters are homogeneous,which is available for targeting for breast cancer cells in vitro.展开更多
To investigate the phenotypic knockout of HIV-1 chemokine coreceptor CXCR4 and CCR5 by intrakines and its inhibitory effect on HIV-1 infection. Primary human PBLs were transduced with the recombinant vector pLNCX-R-K-...To investigate the phenotypic knockout of HIV-1 chemokine coreceptor CXCR4 and CCR5 by intrakines and its inhibitory effect on HIV-1 infection. Primary human PBLs were transduced with the recombinant vector pLNCX-R-K-S-K followed by anti-NGFR/anti-IgG-magnetic bead method selection and FCM detection. The transduced PBI.S were infected with DP1 HFV-1 virus thereafter envelope-mediated syncytium formation and p24 detection were carried out to study the blockage of HIV-1 infection by co-inactivation of CCR5 and CXCR4. pLNCX-R-K-S-K -transduced PBLs were isolated with an anti-NGFR/anti-IgG-magnetic bead method. After isolation, about 70% of the PBI.S were posi- tive for the NGFR marker. When the transduced PBLs were infected with DP1 HIV-1 virus, envelop-mediated syncytium for- mation was almost completely inhibited by pLNCX-R-K-S-K transfection. Also, p24 antigen was very low in the cultures of pLNCX-R-K-S-K transduced PBLs. pLNCX-R-K-S-K transduction inhibited the produc- tion of DP1 p24 antigen by 15%, 43% and 19% on days 4, 7 and 10 respectively. The lymphocytes with the phenotypic knockout of CCR5 and CXCR4 could protect primary human PBLs from DP1 HIV-1 virus infection.展开更多
基金This work was supported by grants from the National Natural Science Foundation of China(30170270)
文摘Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface.Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 SDF-1KDEL,pcDNA3.1 SDF-154KDEL,pEGFPSDF-1KDEL and pEGFPSDF-154KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate,they were transferred into COS-7 cells with liposome . The exogenous expressions were observed, fusion protein SDF-1His and SDF-154His were confirmed by Western blot,and the SDF-1EGFP and SDF-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1 KDELHis and SDF-1 54 KDELHis expressed in COS-7 cells.Subcelluar localization analysis showed that SDF-1KDELEGFP and SDF-154KDELEGFP were located mainly in endoplasmic reticulum.Conclusion:Four expression vectors pcDNA3.1 SDF-1KDEL, pcDNA3.1SDF-154KDEL, pEGFPSDF-1KDEL and pEGFPSDF-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .
基金National Natural Science Foundation of Chinagrant number:30572209
文摘Objective:To prepare folate liposome complexes of recombinant plasmid pEGFP/SDF-1/KDEL wich contained intrakine SDF-1/KDEL gene and to observe its targeting for breast cancer cells.Methods:Reverse phase evaporation method was employed for preparing folate-liposome complexeses of pEGFP/SDF-1/KDEL,so that preparation condition was optimized;and fluorometric method was taken for testing encapsulation efficiency.Particle diameter of liposome was tested by transmission electron microscopy.Complexeses encapsulated with 0.05-0.25 mg/ml calcein were got and incubated with breast cancer cell line MDA-MB-231 cells for 2 to 12 h,then dissolved them with dimethyl sulfoxide,then detected absorbance with microplate reader.Results:The optimized encapsulation condition are as follows:molecular ratio of lecithin and cholesterol was 3:1,rotary speed was 150 r/min under temperature of 43℃,and encapsulation efficiency reached 81%in this experiment.Average liposome particle diameter was 210 nm;8 h after incubation, the intake of MDA-MB-231 cells to 0.25 mg/ml compounds achieved saturation. Conclusion:The pEGFP/SDF-1/KDEL folic acid liposome complexes prepared has a high encapsulating rate,liposome particle diameters are homogeneous,which is available for targeting for breast cancer cells in vitro.
文摘To investigate the phenotypic knockout of HIV-1 chemokine coreceptor CXCR4 and CCR5 by intrakines and its inhibitory effect on HIV-1 infection. Primary human PBLs were transduced with the recombinant vector pLNCX-R-K-S-K followed by anti-NGFR/anti-IgG-magnetic bead method selection and FCM detection. The transduced PBI.S were infected with DP1 HFV-1 virus thereafter envelope-mediated syncytium formation and p24 detection were carried out to study the blockage of HIV-1 infection by co-inactivation of CCR5 and CXCR4. pLNCX-R-K-S-K -transduced PBLs were isolated with an anti-NGFR/anti-IgG-magnetic bead method. After isolation, about 70% of the PBI.S were posi- tive for the NGFR marker. When the transduced PBLs were infected with DP1 HIV-1 virus, envelop-mediated syncytium for- mation was almost completely inhibited by pLNCX-R-K-S-K transfection. Also, p24 antigen was very low in the cultures of pLNCX-R-K-S-K transduced PBLs. pLNCX-R-K-S-K transduction inhibited the produc- tion of DP1 p24 antigen by 15%, 43% and 19% on days 4, 7 and 10 respectively. The lymphocytes with the phenotypic knockout of CCR5 and CXCR4 could protect primary human PBLs from DP1 HIV-1 virus infection.
