RESUMO
We have constructed two related types of multi-cloning mammalian expression vectors. The first, pMPSVEH/HE, carries the promoter of the myeloproliferative sarcoma virus (MPSV). This promoter was found to be stronger than both the SV40 early and the trans-activated human immunodeficiency virus promoters in many cell lines including human and rodent fibroblastoid, lymphoid or myeloid cells. The other, pBEH/HE, carries the simian virus 40 (SV40) early promoter and origin of replication. This offers the possibility of encapsidation in SV40 pseudovirions and subsequent gene transfer into, e.g., hemopoietic cells, via infection. The usefulness of the expression systems was tested with a number of genes and cell lines.
Assuntos
Transformação Celular Viral , Clonagem Molecular , Vetores Genéticos , Transfecção , Animais , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Clonagem Molecular/métodos , HIV/genética , Células HeLa/metabolismo , Humanos , Linfócitos , Plasmídeos , Regiões Promotoras Genéticas , Retroviridae/genéticaRESUMO
The production of glycosylated forms of the human T cell growth factor (interleukin-2, IL-2) has been studied after transfection of a mouse L cell line and a chinese hamster ovary cell line with a plasmid containing the human chromosomal interleukin-2 gene. Both cell lines produced IL-2 constitutively. Based on their behavior in reversed-phase l.c. and their sodium dodecyl sulfate-gel-electrophoresis pattern, human IL-2 protein secreted by L cells showed a similar distribution of glycosylated (Mr 16 500) and nonglycosylated (Mr 14 500) forms as the natural protein secreted by human peripheral lymphocytes, whereas the hamster cell line secreted preponderantly the glycosylated forms. Exoglycosidase digestion of the 16 500 Mr IL-2 protein shifted the gel electrophoretic mobility towards the low-molecular weight form as is true for the natural glycosylated IL-2, which contains the usual tetrasaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-[alpha-NeuAc-(2----6)]-D-GalNAc (IL-2 N2) and the trisaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-D-GalNAc (IL-2 N1) as the major carbohydrate constituents. These results support the applicability of recombinant DNA technology as a tool for studying glycoprotein biosynthesis in mammalian cells.
Assuntos
Genes , Interleucina-2/análogos & derivados , Animais , Sequência de Carboidratos , Carboidratos/análise , Linhagem Celular , Clonagem Molecular , Cricetinae , Cricetulus , DNA Recombinante/metabolismo , Feminino , Glicosídeo Hidrolases , Interleucina-2/biossíntese , Interleucina-2/genética , Cinética , Células L/imunologia , Camundongos , Oligossacarídeos/análise , Ovário , Plasmídeos , Proteínas Recombinantes/metabolismoRESUMO
Expression, ribosomal frameshifting, and proteolytic processing of HIV-1 GAG and POL proteins were investigated in heterologous mammalian cells in order to elucidate the influence of the cellular background on these events. DNA fragments encoded by the gag and pol region were expressed in two rodent cell lines, LTK- and BHK. Both stably transfected cell lines continuously produce recombinant proteins which react with HIV-specific antisera. The GAG precursor and a 39-kDa proteolytic fragment thereof were the major recombinant proteins detected. Expression of the gag-pol region leads to the production of the GAG-POL precursor. Ribosomal frameshifting at the HIV-1 shifty sequence to a typical extent could be positively demonstrated by an enzyme assay. Despite the presence of the viral protease within the GAG-POL precursors, proteolytic processing of the HIV-derived polyproteins was extremely inefficient. The efficiency could not be enhanced by overexpression of the HIV-1 protease encoding region.