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Partial purification and characterization of bovine fibroblast interferon.
1988
Allen G.K. | Grothaus G.D. | Rosenquist B.D.
Bovine fibroblast interferon (BoF-IFN), produced in primary bovine embryonic kidney cell cultures after priming and infection with bluetongue virus, was purified by controlled pore glass (CPG) chromatography to a specific activity of 10(6) U/mg of protein, with 40% recovery of the original activity. The crude IFN was concentrated more than sevenfold during purification. This proved to be a relatively simple, practical method of obtaining sufficient quantities of partially purified natural BoF-IFN for further studies. The CPG-purified BoF-IFN was further concentrated by sequential ultrafiltration and was analyzed by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis (SDS-PAGE). Interferon, recovered from denaturing conditions either by dialysis against phosphate-buffered saline solution or by dilution in cell culture medium containing 10% fetal bovine serum, migrated as a single stainable protein with molecular weight of 21,000 on analytic SDS-PAGE gels. Recovered IFN activity from preparative SDS-PAGE totalled 8.7% of that applied. Attempts to further purify CPG-purified BoF-IFN by zinc chelate affinity chromatography were unsuccessful.
Show more [+] Less [-]Beaded agarose affinity chromatography of bovine fibroblast interferon
1988
Allen, G.K. | Rosenquist, B.D.
Bovine fibroblast interferon (BoF-IFN), produced in bovine embryonic kidney cell cultures by priming and infection with bluetongue virus, was partially purified by controlled pore glass chromatography. The partially purified B0F-IFN then was subjected to beaded agarose affinity chromatography. The IFN eluted by affinity chromatography in 2 distinct fractions-1 after the addition of 1M NaCl and the other one after the addition of 1.5M NaCl containing 50% ethylene glycol. Analysis of fractions by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis revealed a broad molecular weight range (14,900 to 27,900) for the IFN eluted by 1M NaCl, and 2 discrete molecular weight ranges (16,000 to 19,500 and 28,300 to 34,000) for IFN eluted by 1.5M NaCl containing 50% ethylene glycol. The specific activity of the IFN eluted with 1.5M NaCl containing ethylene glycol was 2.85 X 10(6) U/mg of protein, compared with 5.7 X 10(5) U/mg of protein in the controlled pore glass-purified IFN.
Show more [+] Less [-]Partial purification and characterization of bovine fibroblast interferon
1988
Allen, G.K. | Grothaus, G.D. | Rosenquist, B.D.
Bovine fibroblast interferon (BoF-IFN), produced in primary bovine embryonic kidney cell cultures after priming and infection with bluetongue virus, was purified by controlled pore glass (CPG) chromatography to a specific activity of 10(6) U/mg of protein, with 40% recovery of the original activity. The crude IFN was concentrated more than sevenfold during purification. This proved to be a relatively simple, practical method of obtaining sufficient quantities of partially purified natural BoF-IFN for further studies. The CPG-purified BoF-IFN was further concentrated by sequential ultrafiltration and was analyzed by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis (SDS-PAGE). Interferon, recovered from denaturing conditions either by dialysis against phosphate-buffered saline solution or by dilution in cell culture medium containing 10% fetal bovine serum, migrated as a single stainable protein with molecular weight of 21,000 on analytic SDS-PAGE gels. Recovered IFN activity from preparative SDS-PAGE totalled 8.7% of that applied. Attempts to further pruify CPG-purified BoF-IFN by zinc chelate affinity chromatography were unsuccessful.
Show more [+] Less [-]Attachment of Mycoplasma bovoculi to bovine conjunctival epithelium and lung fibroblasts
1988
Salih, B.A. | Rosenbusch, R.F.
A specialized tip structure in some mycoplasmas facilitates their attachment to host cells. Mycoplasma bovoculi strains FS8-7 and M165/69 did not have specialized membrane structure and did not exhibit capsule when stained with ruthenium red and examined by use of transmission electron microscopy. The organisms attached in vitro to bovine lung fibroblasts, with no apparent specialized structure. Attachment to conjunctival epithelium in vivo was observed (after death) in a calf infected with M bovoculi. Close association between M bovoculi and the host cells was noticed. Mycoplasmal cells pretreated with hyperimmune rabbit serum and labeled with protein A-gold complex had gold particles randomly distributed around the membrane. Gold-labeled monoclonal antibodies, M25.5 and M7.3, which were directed against 2 surface antigens of M bovoculi, also were distributed randomly on the mycoplasmal surface as seen in results of double-labeling experiments.
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