Recombinant Human Erythropoietin Proteins Synthesized in Escherichia coli Cells: Effects of Additional Domains on the in vitro and in vivo Activities.

The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use.

[Use of RT-PCR and enzyme immunoassays for the specific diagnosis of Crimean hemorrhagic fever].

The objective of the investigation was to evaluate the efficiency of the RT-PCR kit "AmplySens CHF" produced by InterlabService of the Central Research Institute of Epidemiology and that of the ELISA kits made by the D. I. Ivanovsky Research Institute of Virology for the specific diagnosis of Crimean hemorrhagic fever (CHF). Examination of sera from CHF patients from the Astrakhan Region showed that positive RT-PCR results were observed in 95.2 and 37.5% on days 4-8 and 9-13 after disease onset, respectively; but they were absent on days 13-17. Positive ELISA-IgM results were found in 93% on disease days 6 to 16. A high percentage (78.9%) of positive IgG samples was seen only on days 9-16. Thus, RT-PCR has a marked efficiency in diagnosing CHF until day 8 of illness while ELISA-IgM has it on day 8 or later. ELISA-IgG can be considered to be a confirming rather than compulsory test. The findings suggest that the RT-PCR kit "AmplySens CHF" produced by InterlabService of the Central Research Institute of Epidemiology and that of the ELISA kits made by the D. I. Ivanovsky Research Institute of Virology have a pronounced sensitivity and specificity and a high efficiency when concurrently used to verify CHF in patients.