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Nonviral production of human interleukin-7 in spodoptera frugiperda insect cells as a soluble recombinant protein.

Mirzaei M, Xu Y, Elias CB, Prakash S - J. Biomed. Biotechnol. (2008)

Bottom Line: Because of its strong immunomodulatory effects, hIL-7 may become a valuable supplementary agent for immunotherapeutical treatments in patients with HIV infection or immunodeficiency.This system allows generation of correctly translated and accurately processed heterologous proteins as soluble recombinant proteins.The levels of secreted hIL-7 in a small scale experiment reached a level of 1.7 microg x 1(-1) under serum-free cell culture conditions.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, and Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, 3775 University Street, Montreal, PQ, Canada H3A 2B4.

ABSTRACT
Human interleukin-7 (hIL-7) is a cytokine secreted by the stromal cells of the red marrow. It is important for proliferation during certain stages of B-cell maturation and for T and NK cell survival, development, and homeostasis. It is a critical growth factor for enhancement and recovery of the immune T-cell. Because of its strong immunomodulatory effects, hIL-7 may become a valuable supplementary agent for immunotherapeutical treatments in patients with HIV infection or immunodeficiency. Human IL-7 has previously been produced in various protein expression systems. In this paper, we present an alternative expression system, in Spodoptera frugiperda cells, for the production of hIL-7 using nonlytic vector systems. This system allows generation of correctly translated and accurately processed heterologous proteins as soluble recombinant proteins. Here we report plasmid construction, transfection, and consequent expression of hIL-7 using this nonlytic insect cell expression system. The levels of secreted hIL-7 in a small scale experiment reached a level of 1.7 microg x 1(-1) under serum-free cell culture conditions.

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Related in: MedlinePlus

Viable cell density, total cell density and viability of the Sf9 insect cells infected with different MOI of rbacmid/hIL-7. Different multiplicity of infection (MOI) between 5 to 0.5 was used. Sf9 cells were cultured in shaker flasks and incubated at 27°C with agitation at 115 rpm. Samples were taken at regular intervals (24, 48, 72, and 96) hpi. (a), (b), (c) Infected cells with MOI:5, 1 and 0.5 of the virus stock, respectively. (d) Control (Noninfected insect cells) (n = 3).
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fig7: Viable cell density, total cell density and viability of the Sf9 insect cells infected with different MOI of rbacmid/hIL-7. Different multiplicity of infection (MOI) between 5 to 0.5 was used. Sf9 cells were cultured in shaker flasks and incubated at 27°C with agitation at 115 rpm. Samples were taken at regular intervals (24, 48, 72, and 96) hpi. (a), (b), (c) Infected cells with MOI:5, 1 and 0.5 of the virus stock, respectively. (d) Control (Noninfected insect cells) (n = 3).

Mentions: Wecompared productivity of hIL-7 in Sf9 cells using BEVS. To infect cells,different multiplicity of infection was used. As shown in Figure 7, the final viable cell density of the uninfected cells (control)was much higher than those of cellsinfected by recombinant virus. The number of viable cells increased to morethan 10 × 106 cells/mL by 96 hours in the control and decreased toless than 1 × 106 cells/mL in cells infected with the virus. Inaddition, the viability of infected cells decreased after 48 hpi, to less than 60%. There was no significant difference inviability between control cells over a period of 96 hpi. The Western blot datashown in Figure 8 indicate that hIL-7 is produced and processed normally in theBEVS system to generate a protein with a similar molecular weight to thatproduced in mammalian cells. The protein was found in both the supernatant and the cell lysates, however, the intracellular protein was found to have multiple molecularweights. These different molecular forms are likely a result of incompleteglycosylation which may occur in this system.


Nonviral production of human interleukin-7 in spodoptera frugiperda insect cells as a soluble recombinant protein.

Mirzaei M, Xu Y, Elias CB, Prakash S - J. Biomed. Biotechnol. (2008)

Viable cell density, total cell density and viability of the Sf9 insect cells infected with different MOI of rbacmid/hIL-7. Different multiplicity of infection (MOI) between 5 to 0.5 was used. Sf9 cells were cultured in shaker flasks and incubated at 27°C with agitation at 115 rpm. Samples were taken at regular intervals (24, 48, 72, and 96) hpi. (a), (b), (c) Infected cells with MOI:5, 1 and 0.5 of the virus stock, respectively. (d) Control (Noninfected insect cells) (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2568039&req=5

fig7: Viable cell density, total cell density and viability of the Sf9 insect cells infected with different MOI of rbacmid/hIL-7. Different multiplicity of infection (MOI) between 5 to 0.5 was used. Sf9 cells were cultured in shaker flasks and incubated at 27°C with agitation at 115 rpm. Samples were taken at regular intervals (24, 48, 72, and 96) hpi. (a), (b), (c) Infected cells with MOI:5, 1 and 0.5 of the virus stock, respectively. (d) Control (Noninfected insect cells) (n = 3).
Mentions: Wecompared productivity of hIL-7 in Sf9 cells using BEVS. To infect cells,different multiplicity of infection was used. As shown in Figure 7, the final viable cell density of the uninfected cells (control)was much higher than those of cellsinfected by recombinant virus. The number of viable cells increased to morethan 10 × 106 cells/mL by 96 hours in the control and decreased toless than 1 × 106 cells/mL in cells infected with the virus. Inaddition, the viability of infected cells decreased after 48 hpi, to less than 60%. There was no significant difference inviability between control cells over a period of 96 hpi. The Western blot datashown in Figure 8 indicate that hIL-7 is produced and processed normally in theBEVS system to generate a protein with a similar molecular weight to thatproduced in mammalian cells. The protein was found in both the supernatant and the cell lysates, however, the intracellular protein was found to have multiple molecularweights. These different molecular forms are likely a result of incompleteglycosylation which may occur in this system.

Bottom Line: Because of its strong immunomodulatory effects, hIL-7 may become a valuable supplementary agent for immunotherapeutical treatments in patients with HIV infection or immunodeficiency.This system allows generation of correctly translated and accurately processed heterologous proteins as soluble recombinant proteins.The levels of secreted hIL-7 in a small scale experiment reached a level of 1.7 microg x 1(-1) under serum-free cell culture conditions.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, and Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, 3775 University Street, Montreal, PQ, Canada H3A 2B4.

ABSTRACT
Human interleukin-7 (hIL-7) is a cytokine secreted by the stromal cells of the red marrow. It is important for proliferation during certain stages of B-cell maturation and for T and NK cell survival, development, and homeostasis. It is a critical growth factor for enhancement and recovery of the immune T-cell. Because of its strong immunomodulatory effects, hIL-7 may become a valuable supplementary agent for immunotherapeutical treatments in patients with HIV infection or immunodeficiency. Human IL-7 has previously been produced in various protein expression systems. In this paper, we present an alternative expression system, in Spodoptera frugiperda cells, for the production of hIL-7 using nonlytic vector systems. This system allows generation of correctly translated and accurately processed heterologous proteins as soluble recombinant proteins. Here we report plasmid construction, transfection, and consequent expression of hIL-7 using this nonlytic insect cell expression system. The levels of secreted hIL-7 in a small scale experiment reached a level of 1.7 microg x 1(-1) under serum-free cell culture conditions.

Show MeSH
Related in: MedlinePlus