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Therapeutic Strategy for the Prevention of Pseudorabies Virus Infection in C57BL/6 Mice by 3D8 scFv with Intrinsic Nuclease Activity.

Lee G, Cho S, Hoang PM, Kim D, Lee Y, Kil EJ, Byun SJ, Lee TK, Kim DH, Kim S, Lee S - Mol. Cells (2015)

Bottom Line: The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays.Intraperitoneal injection of 5 and 10 μg 3D8 scFv resulted in no detectable toxicity.The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Korea.

ABSTRACT
3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and 10 μg 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 LD50 PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% (5 μg) and 47% (10 μg). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

No MeSH data available.


Related in: MedlinePlus

Purification and catalytic activity test of 3D8 single chain variable fragment (scFv) protein. (A) The pIg20-3D8 scFv vector encodes a secretion signal peptide of bacterial alkaline phosphate (PhoA L.P), heavy chain variable region (VH) and light chain variable region (VL) of the 3D8 scFv antibody, thrombin cleavage site, and protein A of Staphylococcus aureus under control of the T7 promoter. The VH and VL chains are joined by a flexible peptide linker (GGGGS)3. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on 10% polyacrylamide gel to identify 3D8 scFv and stained with Coomassie Blue. The arrow is the 3D8 scFv protein (32 kDa). Lane M: molecular weight marker. (C) The BSA and purified endotoxin-free 3D8 scFv protein (0.2 μg) was mixed with 0.25 μg of substrate (ssDNA, dsDNA, ssRNA, and dsRNA). A catalytic activity assay was conducted dependent on reaction time (0, 1, 2, 3, 4, and 5 h). Collected samples showed a degradation pattern following agarose gel electrophoresis.
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f1-molce-38-9-773: Purification and catalytic activity test of 3D8 single chain variable fragment (scFv) protein. (A) The pIg20-3D8 scFv vector encodes a secretion signal peptide of bacterial alkaline phosphate (PhoA L.P), heavy chain variable region (VH) and light chain variable region (VL) of the 3D8 scFv antibody, thrombin cleavage site, and protein A of Staphylococcus aureus under control of the T7 promoter. The VH and VL chains are joined by a flexible peptide linker (GGGGS)3. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on 10% polyacrylamide gel to identify 3D8 scFv and stained with Coomassie Blue. The arrow is the 3D8 scFv protein (32 kDa). Lane M: molecular weight marker. (C) The BSA and purified endotoxin-free 3D8 scFv protein (0.2 μg) was mixed with 0.25 μg of substrate (ssDNA, dsDNA, ssRNA, and dsRNA). A catalytic activity assay was conducted dependent on reaction time (0, 1, 2, 3, 4, and 5 h). Collected samples showed a degradation pattern following agarose gel electrophoresis.

Mentions: E. coli strain BL21 DE3 (pLysE) cells containing plg20-3D8 were grown in 1 L of LB broth with 100 μg/ml ampicillin and 20 μg/ml chloramphenicol at 37°C to express the recombinant 3D8 scFv protein (Fig. 1A). When the A600 of the culture reached 0.8, IPTG (0.5 mM) was added to induce protein expression. The culture supernatant was obtained by centrifugation at 10,000 × g for 10 min at 4°C and filtered through a 0.45-nm membrane. The 3D8 scFv protein was purified from the filtered supernatant using an IgG-Sepharose column (Amersham Pharmacia, USA). The column was washed with 20 bed volumes of PBS and then with two volumes of 5 mM ammonium acetate (pH 5.0). The 3D8 scFv protein was eluted with 0.1 M acetic acid (pH 3.4) in fractions of 1.5 ml each. The eluate in the fractions was neutralized to pH 7.0 with 0.1 volume of 1 M Tris-base (pH 9.5). The 3D8 scFv protein was analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Then, endotoxin contents were determined by Limulus Amebocyte Lysate (LAL) (PYROGENT™ 25 single tests 0.125 EU/ml sensitivity, Lonza, Switzerland). The LAL assay was performed in pyrogen-free tubes which 0.1 ml of 3D8 scFv protein (amount range from 2.5 ug to 100 ug) and LAL reagent were added. After 1 h incubation at 37°C, the tubes were observed by vertical inversion whether a stable solid clot was present or not. The visible solid clot was not observed in test tubes which 3D8 scFv protein added (The values of 3D8 scFv endotoxicity was < 0.125 EU ml−1).


Therapeutic Strategy for the Prevention of Pseudorabies Virus Infection in C57BL/6 Mice by 3D8 scFv with Intrinsic Nuclease Activity.

Lee G, Cho S, Hoang PM, Kim D, Lee Y, Kil EJ, Byun SJ, Lee TK, Kim DH, Kim S, Lee S - Mol. Cells (2015)

Purification and catalytic activity test of 3D8 single chain variable fragment (scFv) protein. (A) The pIg20-3D8 scFv vector encodes a secretion signal peptide of bacterial alkaline phosphate (PhoA L.P), heavy chain variable region (VH) and light chain variable region (VL) of the 3D8 scFv antibody, thrombin cleavage site, and protein A of Staphylococcus aureus under control of the T7 promoter. The VH and VL chains are joined by a flexible peptide linker (GGGGS)3. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on 10% polyacrylamide gel to identify 3D8 scFv and stained with Coomassie Blue. The arrow is the 3D8 scFv protein (32 kDa). Lane M: molecular weight marker. (C) The BSA and purified endotoxin-free 3D8 scFv protein (0.2 μg) was mixed with 0.25 μg of substrate (ssDNA, dsDNA, ssRNA, and dsRNA). A catalytic activity assay was conducted dependent on reaction time (0, 1, 2, 3, 4, and 5 h). Collected samples showed a degradation pattern following agarose gel electrophoresis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588720&req=5

f1-molce-38-9-773: Purification and catalytic activity test of 3D8 single chain variable fragment (scFv) protein. (A) The pIg20-3D8 scFv vector encodes a secretion signal peptide of bacterial alkaline phosphate (PhoA L.P), heavy chain variable region (VH) and light chain variable region (VL) of the 3D8 scFv antibody, thrombin cleavage site, and protein A of Staphylococcus aureus under control of the T7 promoter. The VH and VL chains are joined by a flexible peptide linker (GGGGS)3. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on 10% polyacrylamide gel to identify 3D8 scFv and stained with Coomassie Blue. The arrow is the 3D8 scFv protein (32 kDa). Lane M: molecular weight marker. (C) The BSA and purified endotoxin-free 3D8 scFv protein (0.2 μg) was mixed with 0.25 μg of substrate (ssDNA, dsDNA, ssRNA, and dsRNA). A catalytic activity assay was conducted dependent on reaction time (0, 1, 2, 3, 4, and 5 h). Collected samples showed a degradation pattern following agarose gel electrophoresis.
Mentions: E. coli strain BL21 DE3 (pLysE) cells containing plg20-3D8 were grown in 1 L of LB broth with 100 μg/ml ampicillin and 20 μg/ml chloramphenicol at 37°C to express the recombinant 3D8 scFv protein (Fig. 1A). When the A600 of the culture reached 0.8, IPTG (0.5 mM) was added to induce protein expression. The culture supernatant was obtained by centrifugation at 10,000 × g for 10 min at 4°C and filtered through a 0.45-nm membrane. The 3D8 scFv protein was purified from the filtered supernatant using an IgG-Sepharose column (Amersham Pharmacia, USA). The column was washed with 20 bed volumes of PBS and then with two volumes of 5 mM ammonium acetate (pH 5.0). The 3D8 scFv protein was eluted with 0.1 M acetic acid (pH 3.4) in fractions of 1.5 ml each. The eluate in the fractions was neutralized to pH 7.0 with 0.1 volume of 1 M Tris-base (pH 9.5). The 3D8 scFv protein was analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Then, endotoxin contents were determined by Limulus Amebocyte Lysate (LAL) (PYROGENT™ 25 single tests 0.125 EU/ml sensitivity, Lonza, Switzerland). The LAL assay was performed in pyrogen-free tubes which 0.1 ml of 3D8 scFv protein (amount range from 2.5 ug to 100 ug) and LAL reagent were added. After 1 h incubation at 37°C, the tubes were observed by vertical inversion whether a stable solid clot was present or not. The visible solid clot was not observed in test tubes which 3D8 scFv protein added (The values of 3D8 scFv endotoxicity was < 0.125 EU ml−1).

Bottom Line: The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays.Intraperitoneal injection of 5 and 10 μg 3D8 scFv resulted in no detectable toxicity.The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Korea.

ABSTRACT
3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and 10 μg 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 LD50 PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% (5 μg) and 47% (10 μg). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

No MeSH data available.


Related in: MedlinePlus