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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

Construction of the pIE1/153A (V4) expression plasmid carrying the hIL-7 gene. The hIL-7 gene from pORF9-hIL07 vector was inserted in pIE1/153A(V4) expression plasmid, pIE1/153A(V4)/hIL-7 and pBmA-pac were cotransfected into Sf9 cells. Clones producing hIL-7 were selected using puromycin and analyzed by western blot and SDS page.
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Related In: Results  -  Collection


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fig1: Construction of the pIE1/153A (V4) expression plasmid carrying the hIL-7 gene. The hIL-7 gene from pORF9-hIL07 vector was inserted in pIE1/153A(V4) expression plasmid, pIE1/153A(V4)/hIL-7 and pBmA-pac were cotransfected into Sf9 cells. Clones producing hIL-7 were selected using puromycin and analyzed by western blot and SDS page.

Mentions: The hIL-7 gene was obtained by PCR reaction and digestion with NotI and BamHI enzymesfrom pORF9-hIL07 transfer vector, which were used to facilitate the insertionof the gene into the cut pIE1/153A (V4) or pIZ/V5-His expression vectorplasmids. Cloned plasmids were then transferred to competent E.coli cells, purified according to the manufacturer's protocol, and used fortransfection of Sf9 cells. Figure 1 illustrates the construction of pIE1/153A(V4) expression plasmid carrying hIL-7 gene (pIE1/153A.hIL-7) and thetransfection procedure of the constructed plasmid into the Sf9 insect cells.For transfection, pIE1/153A.hIL-7 plasmid was cotransfectedwith plasmid pBmA.pac containing puromycinresistance gene into the insect cells. The expression characteristics ofthe recombinant hIL-7 protein were analyzed by western blotting using hIL-7specific antibody, as presented in Figure 2, which shows preliminary resultsfor screening of positive clones. In Sf9 insect cells, production andprocessing of hIL-7 proceeded normally, resulting in a protein with molecularweight similar to that produced in mammalian cells. The most productivepolyclone (Sf9hIL-7A1) was selected for further study.


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)

Construction of the pIE1/153A (V4) expression plasmid carrying the hIL-7 gene. The hIL-7 gene from pORF9-hIL07 vector was inserted in pIE1/153A(V4) expression plasmid, pIE1/153A(V4)/hIL-7 and pBmA-pac were cotransfected into Sf9 cells. Clones producing hIL-7 were selected using puromycin and analyzed by western blot and SDS page.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Construction of the pIE1/153A (V4) expression plasmid carrying the hIL-7 gene. The hIL-7 gene from pORF9-hIL07 vector was inserted in pIE1/153A(V4) expression plasmid, pIE1/153A(V4)/hIL-7 and pBmA-pac were cotransfected into Sf9 cells. Clones producing hIL-7 were selected using puromycin and analyzed by western blot and SDS page.
Mentions: The hIL-7 gene was obtained by PCR reaction and digestion with NotI and BamHI enzymesfrom pORF9-hIL07 transfer vector, which were used to facilitate the insertionof the gene into the cut pIE1/153A (V4) or pIZ/V5-His expression vectorplasmids. Cloned plasmids were then transferred to competent E.coli cells, purified according to the manufacturer's protocol, and used fortransfection of Sf9 cells. Figure 1 illustrates the construction of pIE1/153A(V4) expression plasmid carrying hIL-7 gene (pIE1/153A.hIL-7) and thetransfection procedure of the constructed plasmid into the Sf9 insect cells.For transfection, pIE1/153A.hIL-7 plasmid was cotransfectedwith plasmid pBmA.pac containing puromycinresistance gene into the insect cells. The expression characteristics ofthe recombinant hIL-7 protein were analyzed by western blotting using hIL-7specific antibody, as presented in Figure 2, which shows preliminary resultsfor screening of positive clones. In Sf9 insect cells, production andprocessing of hIL-7 proceeded normally, resulting in a protein with molecularweight similar to that produced in mammalian cells. The most productivepolyclone (Sf9hIL-7A1) was selected for further study.

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