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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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Growth curve of the stably transfected Sf9 producing hIL-7 and nontransfected Sf9 insect cells. (a)  Viable cell density, total cell density, and viability of stably transfected Sf9 with pIZ/V5-hIL-7 plasmid. Cells were seeded at a density of  5 × 105 cells·mL−1 and maintained at 1 × 107 cells·mL−1. (b) Viable cell density, total cell density, and viability of nontransfected Sf9 insect cells. Cells were seeded at a density of 5 × 105 cells·mL−1 (n = 3).
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fig4: Growth curve of the stably transfected Sf9 producing hIL-7 and nontransfected Sf9 insect cells. (a) Viable cell density, total cell density, and viability of stably transfected Sf9 with pIZ/V5-hIL-7 plasmid. Cells were seeded at a density of 5 × 105 cells·mL−1 and maintained at 1 × 107 cells·mL−1. (b) Viable cell density, total cell density, and viability of nontransfected Sf9 insect cells. Cells were seeded at a density of 5 × 105 cells·mL−1 (n = 3).

Mentions: For comparison, wecloned the nucleotide sequences corresponding to the hIL-7 protein into the pIZ/V5-Hisexpression vector and constructed the pIZ/V5-His.hIL-7vector. This plasmid is an immediate early expressionplasmid where the coding sequence for hIL-7 is positioned under thetranscription control of the OpIE2 promoter. The promoter is derivedfrom a second immediate early regulatory gene of the baculovirus Orgyia pseudotsugata multicapsid nuclearpolyhedrosis virus (OpMNPV) which was identified in 1992 [20, 22]. Insect cells were then transfected with pIZ/V5-His.hIL-7and stable transfectants were selected with zeocin, aglycopeptide antibiotic of the bleomycin family, which is active in vivo against most bacteria(including E. coli), eukaryotic microorganisms (i.e., yeasts), plantcells, and animal cells [19]. Themost productive polyclonals, grown with Sf900IImedium in shake flasks at 27°C and 120 rpm from an initial concentration of 5 × 105 cell·mL−1, reached a maximum celldensity of approximately 8 × 106 cell/mL on the fourthday of culture with a cell viability of 93% (Figure 4(a)). This demonstrated thatstable transfection of pIZ/V5-His.hIL-7 into Sf9 cells did not cause significant changes in thegrowth of the transfected Sf9-hIL-7 cells nor in the cell viability, whencompared to growth curves of wild-type Sf9 cells obtained under the sameconditions (Figure 4(b)).


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)

Growth curve of the stably transfected Sf9 producing hIL-7 and nontransfected Sf9 insect cells. (a)  Viable cell density, total cell density, and viability of stably transfected Sf9 with pIZ/V5-hIL-7 plasmid. Cells were seeded at a density of  5 × 105 cells·mL−1 and maintained at 1 × 107 cells·mL−1. (b) Viable cell density, total cell density, and viability of nontransfected Sf9 insect cells. Cells were seeded at a density of 5 × 105 cells·mL−1 (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2568039&req=5

fig4: Growth curve of the stably transfected Sf9 producing hIL-7 and nontransfected Sf9 insect cells. (a) Viable cell density, total cell density, and viability of stably transfected Sf9 with pIZ/V5-hIL-7 plasmid. Cells were seeded at a density of 5 × 105 cells·mL−1 and maintained at 1 × 107 cells·mL−1. (b) Viable cell density, total cell density, and viability of nontransfected Sf9 insect cells. Cells were seeded at a density of 5 × 105 cells·mL−1 (n = 3).
Mentions: For comparison, wecloned the nucleotide sequences corresponding to the hIL-7 protein into the pIZ/V5-Hisexpression vector and constructed the pIZ/V5-His.hIL-7vector. This plasmid is an immediate early expressionplasmid where the coding sequence for hIL-7 is positioned under thetranscription control of the OpIE2 promoter. The promoter is derivedfrom a second immediate early regulatory gene of the baculovirus Orgyia pseudotsugata multicapsid nuclearpolyhedrosis virus (OpMNPV) which was identified in 1992 [20, 22]. Insect cells were then transfected with pIZ/V5-His.hIL-7and stable transfectants were selected with zeocin, aglycopeptide antibiotic of the bleomycin family, which is active in vivo against most bacteria(including E. coli), eukaryotic microorganisms (i.e., yeasts), plantcells, and animal cells [19]. Themost productive polyclonals, grown with Sf900IImedium in shake flasks at 27°C and 120 rpm from an initial concentration of 5 × 105 cell·mL−1, reached a maximum celldensity of approximately 8 × 106 cell/mL on the fourthday of culture with a cell viability of 93% (Figure 4(a)). This demonstrated thatstable transfection of pIZ/V5-His.hIL-7 into Sf9 cells did not cause significant changes in thegrowth of the transfected Sf9-hIL-7 cells nor in the cell viability, whencompared to growth curves of wild-type Sf9 cells obtained under the sameconditions (Figure 4(b)).

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