OBJECTIVE The FLT-3 ligand （fms-like tyrosine kinase receptor-3 ligand, FL） is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentia...OBJECTIVE The FLT-3 ligand （fms-like tyrosine kinase receptor-3 ligand, FL） is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentiation of primitive hematopoietic cells. To yield a high-level of recombinant human FL protein, a recembinant Pichia Pastoris （P. pastoris）strain was constructed. METHODS An artificial expression frame, with the same encoding protein sequence for the FL extracellular domain cDNA, was synthesized by using favored genetic codons of P. pastoris. P. pastoris strain KM71 cells were transformed with the endonuclease Bgl II linearized recombined plasmid, pPIC9K-FL. The plasmid then was linerized in the 5＇AOX1 site and integrated into the yeast KM71 genome. KM71 was transformed with pPIC9K plasmids as a control for the production of recombinant protein. Southern blotting and Northern blotting tests were used to screen the genotype of the recombined strain. Biological activity was demonstrated in vitro with culturing of CD34＋cells. RESULTS The recombinant human FL protein expressed into the yeast culture supertant was identified on the basis of its molecular weight and Western blotting analysis. Numerous bands were observed in the 10-100 kDa molecular mass range. SDS-PAGE showed that the expressed product, a 20 kDa protein, was secreted into the medium in the form of a soluble molecule. Western-blot analyses showed good antigenicity and specificity against polyclonal antibodies. A sharp band and a smeared band were observed at a molecular mass of approximately 20 kDa by Western blotting. The recombinant human FL protein was the major protein component observed in the culture supernatant. The highest yield （108 mg/L） was obtained when expression was induced with 0.5% methanol for 96 h. Deglycosylation with PNGase F resulted in a decrease in apparent molecular mass from 20 kDa to 18kDa forming three bands all of which were also detected by rabbit anti-FL antibodies, Culturing of CD34＋ cells in the presence of KM71pPIC9K-FL over 7 days increased 2.9 fold, while in the control group they increased only 1,5 fold. The biological assay showed that the expressed product could stimulate the proliferation of CD34＋ hematopoietic cells, CONCLUSION We demonstrated that human FL was secreted into the culture supernatant from P. pastoris, and that this yeast strain was a preferred host for recombinant human FL gene expression. This recombinant strain can provide a convenient process for pharmaceutical application.展开更多
Objective: To investigate the F1t3 receptor expression on the surface of malignant hematopoietic cells, the effect of TNFα and dexamethasone (DXM) on its expression and the responses of those cells to recombinant hum...Objective: To investigate the F1t3 receptor expression on the surface of malignant hematopoietic cells, the effect of TNFα and dexamethasone (DXM) on its expression and the responses of those cells to recombinant human F1t3 ligand (rhFL). Methods: Eighteen malignant hematopoietic cell lines were determined for the F1t3 receptor expression by flow cytometric analysis. The effect of rhFL on the proliferation of malignant hematopoietic cellsin vitro was measured using MTT assay. Results: The expressions of F1t3 receptor on the surface of Raji, Daudi, HL-60, 8266 and XG-6 cells were detected by flow cytometric analysis. Following incubation with 20 ng/ml TNFα for 24h, the number of F1t3 receptor positive cells decreased in Raji and 8266, increased in HL-60 and XG-6, and no difference in Daudi cells. After incubation with 10?6 mol/L DXM for 24h, the number of F1t3 receptor positive cells decreased in all the 5 F1t3 receptor positive cell lines. rhFL stimulated the proliferation of HL-60 and Raji cells. Conclusion: For most of the malignant hematopoietic cells, there was neither the expression of F1t3 receptor nor the response to rhFL. DXM may be useful to reduce the effect of FL on the proliferation of some F1t3 receptor positive malignant hematopoietic cells in vitro andin vivo.展开更多
文摘OBJECTIVE The FLT-3 ligand （fms-like tyrosine kinase receptor-3 ligand, FL） is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentiation of primitive hematopoietic cells. To yield a high-level of recombinant human FL protein, a recembinant Pichia Pastoris （P. pastoris）strain was constructed. METHODS An artificial expression frame, with the same encoding protein sequence for the FL extracellular domain cDNA, was synthesized by using favored genetic codons of P. pastoris. P. pastoris strain KM71 cells were transformed with the endonuclease Bgl II linearized recombined plasmid, pPIC9K-FL. The plasmid then was linerized in the 5＇AOX1 site and integrated into the yeast KM71 genome. KM71 was transformed with pPIC9K plasmids as a control for the production of recombinant protein. Southern blotting and Northern blotting tests were used to screen the genotype of the recombined strain. Biological activity was demonstrated in vitro with culturing of CD34＋cells. RESULTS The recombinant human FL protein expressed into the yeast culture supertant was identified on the basis of its molecular weight and Western blotting analysis. Numerous bands were observed in the 10-100 kDa molecular mass range. SDS-PAGE showed that the expressed product, a 20 kDa protein, was secreted into the medium in the form of a soluble molecule. Western-blot analyses showed good antigenicity and specificity against polyclonal antibodies. A sharp band and a smeared band were observed at a molecular mass of approximately 20 kDa by Western blotting. The recombinant human FL protein was the major protein component observed in the culture supernatant. The highest yield （108 mg/L） was obtained when expression was induced with 0.5% methanol for 96 h. Deglycosylation with PNGase F resulted in a decrease in apparent molecular mass from 20 kDa to 18kDa forming three bands all of which were also detected by rabbit anti-FL antibodies, Culturing of CD34＋ cells in the presence of KM71pPIC9K-FL over 7 days increased 2.9 fold, while in the control group they increased only 1,5 fold. The biological assay showed that the expressed product could stimulate the proliferation of CD34＋ hematopoietic cells, CONCLUSION We demonstrated that human FL was secreted into the culture supernatant from P. pastoris, and that this yeast strain was a preferred host for recombinant human FL gene expression. This recombinant strain can provide a convenient process for pharmaceutical application.
文摘Objective: To investigate the F1t3 receptor expression on the surface of malignant hematopoietic cells, the effect of TNFα and dexamethasone (DXM) on its expression and the responses of those cells to recombinant human F1t3 ligand (rhFL). Methods: Eighteen malignant hematopoietic cell lines were determined for the F1t3 receptor expression by flow cytometric analysis. The effect of rhFL on the proliferation of malignant hematopoietic cellsin vitro was measured using MTT assay. Results: The expressions of F1t3 receptor on the surface of Raji, Daudi, HL-60, 8266 and XG-6 cells were detected by flow cytometric analysis. Following incubation with 20 ng/ml TNFα for 24h, the number of F1t3 receptor positive cells decreased in Raji and 8266, increased in HL-60 and XG-6, and no difference in Daudi cells. After incubation with 10?6 mol/L DXM for 24h, the number of F1t3 receptor positive cells decreased in all the 5 F1t3 receptor positive cell lines. rhFL stimulated the proliferation of HL-60 and Raji cells. Conclusion: For most of the malignant hematopoietic cells, there was neither the expression of F1t3 receptor nor the response to rhFL. DXM may be useful to reduce the effect of FL on the proliferation of some F1t3 receptor positive malignant hematopoietic cells in vitro andin vivo.
