Production of recombinant human microplasminogen and pilot study in inducing posterior vitreous detachment.
Curr Eye Res
; 30(10): 881-9, 2005 Oct.
Article
em En
| MEDLINE
| ID: mdl-16251125
ABSTRACT
PURPOSE:
To realize the high production of recombinant human microplasminogen (r-mPlg) with Pichia pastoris and demonstrate the efficacy of r-mPlg in inducing posterior vitreous detachment (PVD).METHODS:
Recombinant plasmid pAO815-3mPlg was constructed and transformed into SMD1168 cells. Positive recombinant clones were selected with MD plate and cultured in BMG medium, then induced in BMM medium. A protein band corresponding to mPlg with molecular mass of 29 kDa was revealed in SDS-PAGE and confirmed by Western blot. Anion-exchange chromatography and plasminogen activity assay kit were used to obtain purified r-mPlg with biological activity. Twenty eyes of freshly slaughtered pigs were divided into 4 groups, 5 eyes in each group. Group 1 served as normal control. Intravitreal injection of 0.1 ml BSS, 1000 IU/0.1 ml recombinant streptokinase (r-SK) and 1000 IU/0.1 ml r-SK plus 3 U/0.1 ml r-mPlg was done respectively to groups 2, 3, and 4. After incubation at 37 degrees C for 60 min, all eyes were processed for light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM).RESULTS:
r-mPlg, which has potential fibrinolytic activity, was successfully obtained with yield of 30 mg/L and purity of 97%. PVD was demonstrated by SEM in group 4 but not in other three groups. The retina and the inner limiting membrane (ILM) were well preserved in all eyes.CONCLUSION:
r-mPlg, which has potential fibrinolytic activity, can be produced through Pichia pastoris expression system. Three U of r-mPlg combined with 1000 IU r-SK was effective in producing PVD without damaging the retina.
Buscar no Google
Base de dados:
MEDLINE
Assunto principal:
Fragmentos de Peptídeos
/
Plasminogênio
/
Descolamento do Vítreo
Idioma:
En
Ano de publicação:
2005
Tipo de documento:
Article