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Prokaryotic expression and purification of fusion protein HSP70-EGFP and its application in the study

QU Ping, SUI Yanfang, MA Jiahai, CHEN Guangsheng, LIU Libing, CHEN Jiankang, LIU Fang'e

《医学前沿（英文）》 2008年第2卷第1期页码 82-86 doi: 10.1007/s11684-008-0015-0

摘要： To study the endocytic activity of dendritic cells (DCs) by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood. Fusion protein HSP70-EGFP was prokaryotically expressed, isolated and purified. DCs were isolated and cultured from human peripheral blood. The DCs were divided into 3 groups in the endocytic experiment. There were 10 DCs in each group. Group 1 and 2 were respectively incubated for 30 min. with HSP70-EGFP and EGFP. Group 3 was incubated with HSP70 for 30 min, and then incubated for 30 min. with HSP70-EGFP. Subsequently, 3 groups were placed in an incubator at 37°C for 0.5, 1, 2 and 24 h. Flow cytometry (FCM) was adopted to detect the amount of DCs with EGFP inside. IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12. There were 5 types in the experiment: LPS, inactive LPS, HSP70-EGFP, EGFP and no antigen. Fusion protein HSP70-EGFP was successfully obtained and its molecular weight was 97 000. It accounted for 35.32% of the total protein. Under irradiation of an ultraviolet lamp, the protein solution sent out viridescent fluorescence. The result detected by FCM indicated that after incubation for 0.5 h at 37°C, the positive rate in group 1 was 63%, while the other 2 groups were negative. After incubation for 1, 2 and 24 h at 37°C, the positive rates in the 3 groups were above 80%. The IL-12 Eli-spot examination shows that with HSP70-EGFP being loaded, the amount of DCs secreting IL-12 was 134.09 ± 31.78/10 cells, a little lower than that of DCs with LPS loaded (with the average point of 156.36 ± 15.73). There was no significant difference between the 2 groups ( < 0.01). By contrast, both of them were significantly higher than inactive LPS-(33.78 ± 1.40)/10 cells and EGFP-loaded (23.13 ± 4.57)/10 cells DC groups in the amount of DCs secreting IL-12 ( < 0.01). The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs internalizing HSP70-EGFP. With increasing incubation time, pinocytosis begins to dominate. HSP70-EGFP may promote DCs to secret cell factor IL-12.

关键词： significant difference irradiation peripheral preliminary EGFP

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Effect of calcium on porcine ICSI embryos expressing EGFP is related to activation of ooplasmic DNase

Shuaishuai WU,Heng CHEN,Yingzheng WANG,Hui GAO,Shenming ZENG

《农业科学与工程前沿（英文）》 2015年第2卷第1期页码 84-89 doi: 10.15302/J-FASE-2015046

摘要： Several reliable methods to produce transgenic animals use the male genome. After penetration into oocytes, sperm DNA undergoes dramatic conformational changes that might represent an opportunity for exogenous DNA to integrate into the zygote genome. A nuclease, DNase I, with Ca /Mg dependent activity and Zn inhibition, is one of the enzymes responsible for sperm DNA remodeling. To date, the effect of different calcium concentrations in manipulation media on porcine intracytoplasmic sperm injection has not been fully investigated. The present study was conducted to examine the effect of calcium in the surrounding media, and we found that the number of embryos expressing green fluorescent protein (EGFP) was increased in media containing Ca . However, the number did not change over Ca concentrations from 2 to 8 mmol·L ( >0.05). Moreover, free Ca concentrations in the media were found to affect the efficiency which is ICSI embryos expressing EGFP protein, which was related to the activation of ooplasmic DNase I. These findings reveal a mechanism and pathway involving EGFP expression in ICSI embryos.

关键词： ICSI calcium DNase I GFP porcine