RESUMO
The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.
Assuntos
Fator VII/biossíntese , Leishmania/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Animais , Testes de Coagulação Sanguínea , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Fator VII/química , Fator VII/genética , Células Hep G2 , Humanos , Leishmania/citologia , Leishmania/genética , Lagartos/parasitologia , Modelos Moleculares , Plasmídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
OBJECTIVES: To evaluate human herpesvirus 8/Kaposi's sarcoma associated herpesvirus (HHV-8/KSHV) viral load in diagnostic, (formalin fixed, paraffinised) biopsies and patient serum during tumour progression of oral and cutaneous AIDS-related Kaposi's sarcoma (AKS), and endemic Kaposi's sarcoma (EKS) by a sensitive and specific quantitative real time polymerase chain reaction (qRT-PCR) assay. STUDY DESIGN: Eighty six biopsies of both AKS (oral and cutaneous AKS, 68) and EKS (cutaneous EKS, 18) were evaluated by qRT-PCR and immunohistochemistry (IHC). The viral load in human tumour tissue and serum of some individual patients were compared. RESULTS: Higher viral load as well as frequency of latency-associated nuclear antigen (LANA)+ tumour spindle cells (SC) and number of LANA granules per SC was found in oral AKS compared to cutaneous AKS. Although few cases were available, serum viral load appeared to decrease compared to tumour tissue during KS progression. CONCLUSIONS: The higher viral load in oral rather than cutaneous AKS is consistent with the well recognised reservoir function of the oral mucosa. Decrease of serum HHV-8 load during KS progression may indicate decreased virus release and/or increased virus clearance.