RESUMO
We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.
Assuntos
Anticorpos/imunologia , Mapeamento de Epitopos , Radioimunoensaio/métodos , Relaxina/análise , Sequência de Aminoácidos , Estradiol/administração & dosagem , Estradiol/uso terapêutico , Terapia de Reposição de Estrogênios , Feminino , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Gravidez , Conformação Proteica , Relaxina/sangue , Relaxina/imunologia , Relaxina/urina , Sêmen/química , Sensibilidade e EspecificidadeRESUMO
We have investigated large scale production processes (up to 2 liters) of recombinant proteins using the baculovirus expression system in order to optimize the product yields. Experiments using cell lines of Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-Mb0503) were performed to show the different production capacities of the cell lines. The influence of the infection at different cell densities is described. Beyond that, TC100-, IPL41- and serum-free IPL41-medium were compared to demonstrate their different capabilities of supporting cell growth and protein expression. Additionally, the inhibitory effect of FCS on the protease activity of kallikrein, which is produced in its zymogenic form, is discussed Improved production parameters are described, which enabled us to produce up to 8000 units of activated pro-kallikrein within 14 days using perfusion cultivation.
Assuntos
Baculoviridae/metabolismo , Biotecnologia , Precursores Enzimáticos/biossíntese , Calicreínas/biossíntese , Animais , Bovinos , Contagem de Células , Células Cultivadas , Meios de Cultura , Precursores Enzimáticos/metabolismo , Humanos , Calicreínas/metabolismo , Mariposas/citologia , Mariposas/metabolismo , Perfusão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , TripsinaRESUMO
A cDNA fragment encoding human salivary-gland kallikrein, including the kallikrein-owned signal peptide, was inserted into a baculovirus vector adjacent to the polyhedrin promoter and expressed in transfected insect cells. Biologically active kallikrein was isolated to homogeneity from serum-free culture supernatant using a four-step protocol. The N-terminal amino acid sequence of the insect-derived kallikrein was identical to that of the natural proteinase, thus indicating the proper removal of the mammalian signal peptide.
Assuntos
Calicreínas/genética , Glândulas Salivares/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Linhagem Celular , Expressão Gênica , Vetores Genéticos , Insetos , Calicreínas/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The aim of our study was to establish an efficient system for the in vitro production of the insect pathogenic Autographa californica nuclear polyhedrosis virus in a Spodoptera frugiperda cell line. We optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. Cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. In order to provide sufficient nutrients during virus synthesis filtration and perfusion devices were connected to the bioreactor. A virus production procedure in a repeated batch mode by using a two stage bioreactor system is described. Stage I was optimized for cell production and stage II for virus production.
Assuntos
Baculoviridae/crescimento & desenvolvimento , Técnicas Citológicas , Cultura de Vírus/métodos , Animais , Biotecnologia , Linhagem Celular , Meios de Cultura , Insetos , OxigênioRESUMO
Cell cycle kinetic of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G0) of mammalian cells is characterized by a 2c-DNA content whereas G0-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2'-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.
