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Prokaryotic expression and purification of fusion
protein HSP70-EGFP and its application in the study of dendritic cells
internalization antigen |
| QU Ping1, SUI Yanfang2, MA Jiahai2, CHEN Guangsheng2, LIU Libing3, CHEN Jiankang3, LIU Fang'e3 |
| 1.Department of Pathology, School of Basic Medicine, Fourth Military Medical University; Center of Basic Medicine and Teaching Experiment, School of Basic Medicine, Fourth Military Medical University; 2.Department of Pathology, School of Basic Medicine, Fourth Military Medical University; 3.Center of Basic Medicine and Teaching Experiment, School of Basic Medicine, Fourth Military Medical University |
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Abstract 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 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°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 ± 31.78/105 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 internalizing 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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Issue Date: 05 March 2008
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| 1 |
Wallin R P Lundqvist A Moré S H von Bonin A Kiessling R Ljunggren H G Heat-shock proteins as activators of the innate immunesystemTrends Immunol 2002 23(3)130135
|
| 2 |
Kol A Lichtman A H Finberg R W Libby P Kurt-Jones E A Cutting edge: heat shock protein(HSP)60activates the innate immune response: CD14 is an essential receptorfor HSP60 activation of mononuclear cellsJ Immunol 2000 164(1)1317
|
| 3 |
Qu P Sui Y F Ye J Zhang X M Gene clone,expression and purification of enhanced green fluorescent proteinShanxi Yixue Zazhi 2006 35(10)12351237 (in Chinese)
|
| 4 |
Zhang J F Yu W B Xu X L Su M Q Ma Y Y Liu J Y Ding Z R In vitro generation of human blood dendritic cells for active tumor imm unotherapyDi Si Junyi Daxue Xuebao 2001 22(19)17771780 (in Chinese)
|
| 5 |
Li Z Qiao Y Liu B Laska E J Chakravarthi P Kulko J M Bona R D Fang M Hegde U Moyo V Tannenbaum S H Ménoret A Gaffney J Glynn L Runowicz C D Srivastava P K Combination of imatinib mesylatewith autologous leukocyte-derived heat shock protein and chronic myelogenousleukemiaClin Cancer Res 2005 11(12)44604468
|
| 6 |
Ye J Chen G S Song H P Li Z S Huang Y Y Qu P Sun Y J Zhang X M Sui Y F Heat shockprotein 70/MAGE-1 tumor vaccine can enhance the potency of MAGE-1-unspecificcellular immune responses in vivoCancerImmunol Immunother 2004 53(9)825834
|
| 7 |
Zhang H Wang W Li Q Huang W Fusion proteinof ATPase domain of Hsc70 with TRP2 acting as a tumor vaccine againstB16 melanomaImmunol Let 2006 105(2)167173
|
| 8 |
Zhao Z L Yan L Cui F D Shi J G Long G N HSP-peptide complex vaccine in preventingpostoperative recurrence of hepatocarcinoma: 32 cases reportDi Si Junyi Daxue Xuebao 2005 26(22)20762078 (in Chinese)
|
| 9 |
Huang Q Richmond J F Suzue K Eisen H N Young R A In vivo cytotoxic T lymphocyte elicitationby mycobacterial heat shock protein 70 fusion proteins maps to a discretedomain and is CD4(+) T cell independentJ Exp Med 2000 191(2)403408
|
| 10 |
Singh-Jasuja H Toes R E Spee P Münz C Hilf N Schoenberger S P Ricciardi-Castagnoli P Neefjes J Rammensee H G Arnold-Schild D Schild H Cross-presentation of g]ycoprotein96-associated antigens on major histocompatibility complex class Imolecules requires receptor-mediated endocytosisJ Exp Med 2000 191(11)19651974
|
| 11 |
Castellino F Boucher P E Eichelberg K Mayhew M Rothman J E Houghton A N Germain R N Receptor-mediated uptake ofantigen/heat shock protein complexes results in major histocompatibilitycomplex class I antigen presentation via two distinct processing pathwaysJ Exp Med 2000 191(11)19571964
|
| 12 |
Arnold-Schild D Hanau D Spehner D Schmid C Rammensee H G de la Salle H Schild H Cutting edge:Receptor-mediated endocytosis of heat shock proteins by professionalantigen-presenting cellsJ Immunol 1999 162(7)37573760
|
| 13 |
Basu S Binder R J Ramalingam T Srivastava P K CD91is a common receptor for heat shock proteins gp96, hsp90, hsp70, andcalreticulinImmunity 2001 14(3)303313
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