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

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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 puri-fied. DCs were isolated and cultured from human peri-pheral blood. The DCs were divided into 3 groups in the endocytic experiment. There were 106 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℃ 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 pro-tein 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 fluor-escence. The result detected by FCM indicated that after incubation for 0.5 h at 37℃, 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℃, the positive rates in the 3 groups were above 80%. The IL-12 Eli-spot exam-ination shows that with HSP70-EGFP being loaded, the amount of DCs secreting IL-12 was 134.09±31.78/105 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 (P<0.01). By contrast, both of them were significantly higher than inactive LPS-(33.78±1.40)/105 cells and EGFP-loaded (23.13±4,57)/105 cells DC groups in the amount of DCs secreting IL-12 (P<0.01). The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs intealizing HSP70-EGFP. With increasing incubation time, pinocytosis begins to dominate. HSP70-EGFP may promote DCs to secret cell factor IL-12.
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