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
目的 观察HIV 1辅受体CCR5和CXCR4表型剔除对HIV 1DP1株感染的阻断作用。方法 用含有pLNCX R K S K的重组逆转录病毒液感染原代人PBLs(外周血淋巴细胞 ) ,抗体 免疫磁珠法分离筛选转化成功PBLs,流式细胞仪检测筛选效率 ;HIV 1DP1株...目的 观察HIV 1辅受体CCR5和CXCR4表型剔除对HIV 1DP1株感染的阻断作用。方法 用含有pLNCX R K S K的重组逆转录病毒液感染原代人PBLs(外周血淋巴细胞 ) ,抗体 免疫磁珠法分离筛选转化成功PBLs,流式细胞仪检测筛选效率 ;HIV 1DP1株攻击转化PBLs ,进行合胞体形成和p2 4抗原分泌检测。结果 抗 NGFR 免疫磁珠法获得了转化成功的PBLs ,流式细胞仪检测发现pLNCX R K S K转染组 77.4%的PBLsNGFR(神经生长因子受体 )标记物为阳性 ;HIV 1DP1株攻击后 ,未转染组和pLNCX转染组可以见到明显的合胞体形成 ,而在pLNCX R K S K转化PBLs没有见到合胞体形成 ;pLNCX R K S K转染组在感染后第 4、7和 10天皆可发现显著的p2 4抗原分泌抑制 ,抑制率分别为 15%、43 %、19%。结论 细胞内趋化因子通过与CCR5和CXCR4细胞内结合 ,使HIV 1两类主要辅受体难以在PBLs表面表达 ,从而可以达到阻断HIV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.
文摘目的 观察HIV 1辅受体CCR5和CXCR4表型剔除对HIV 1DP1株感染的阻断作用。方法 用含有pLNCX R K S K的重组逆转录病毒液感染原代人PBLs(外周血淋巴细胞 ) ,抗体 免疫磁珠法分离筛选转化成功PBLs,流式细胞仪检测筛选效率 ;HIV 1DP1株攻击转化PBLs ,进行合胞体形成和p2 4抗原分泌检测。结果 抗 NGFR 免疫磁珠法获得了转化成功的PBLs ,流式细胞仪检测发现pLNCX R K S K转染组 77.4%的PBLsNGFR(神经生长因子受体 )标记物为阳性 ;HIV 1DP1株攻击后 ,未转染组和pLNCX转染组可以见到明显的合胞体形成 ,而在pLNCX R K S K转化PBLs没有见到合胞体形成 ;pLNCX R K S K转染组在感染后第 4、7和 10天皆可发现显著的p2 4抗原分泌抑制 ,抑制率分别为 15%、43 %、19%。结论 细胞内趋化因子通过与CCR5和CXCR4细胞内结合 ,使HIV 1两类主要辅受体难以在PBLs表面表达 ,从而可以达到阻断HIV
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.
