RESUMO
We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone.
Assuntos
Replicação do DNA/genética , DNA Recombinante/biossíntese , DNA Recombinante/genética , Engenharia Genética/métodos , Regiões de Interação com a Matriz/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Plasmídeos/genética , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , TransfecçãoRESUMO
A statistical investigation was carried out on the distribution of serum N-carbamoyl-beta-D-glucopyranosylamine (NCG) among various patient groups. The serum NCG levels of patients treated in the departments of hemodialysis (131 +/- microM), nephritic syndrome (47 +/- 54 microM), and diabetes mellitus (55 +/- 70 microM) were significantly higher (p < 0.01) than those in other internal disease patients (18 +/- 22 microM) and healthy volunteers (6 +/- 22 microM). The serum NCG level was greatly reduced by hemodialysis therapy, however a return to initial NCG levels was observed within about one week. These results indicate that a high serum NCG level is a feature of renal failure patients, and a relationship was demonstrated between hyperuremia and NCG formation and accumulation in blood.
Assuntos
Glucosamina/sangue , Insuficiência Renal/sangue , Biomarcadores/sangue , Diabetes Mellitus/sangue , Glucosamina/análogos & derivados , Humanos , Diálise Renal , Insuficiência Renal/terapia , SíndromeRESUMO
We attempted to develop a novel serum 1,5-anhydroglucitol (1,5-AG) assay kit using glucose 3-dehydrogenase (G3DH) from Halomonas sp. alpha-15 strain. The major advantages of this method are that the 1,5-AG detection requires a very small amount of G3DH, and the enzyme catalyzes a simple reaction. The analytical performances were acceptable for clinical use operated with a clinical automatic analyzer. The correlation with a commercial assay kit against sera of healthy volunteers was y=0.975x+0.008, r=0.993, Sylx=1.32 microg/mL. However, sham-negative specimens were observed in the validation of this method using specimens from hospital patients.
