Correction to: Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells.

The original publication has been updated. The acknowledgment was omitted from the original article and is published below.

Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells.

Non-glycosylated, recombinant human granulocyte colony-stimulating factor (rhG-CSF), produced by Escherichia coli (filgrastim, leukostim) is widely used to treat a number of serious human diseases and aids in the recovery post bone marrow transplantation. Although glycosylation is not required for the manifestation of the biological activity of G-CSF, a number of studies have shown that the carbohydrate residue significantly increases the physicochemical stability of the G-CSF molecule. Therefore, the aim of the present study was to design a Pichia pastoris strain capable of producing glycosylated rhG-CSF, and to study its effects on rat bone marrow cells. The nucleotide sequence of the rhG-CSF gene has been optimized for expression in P. pastoris, synthesized, cloned into the pPICZαA vector and expressed under the control of the AOX promoter in P. pastoris X33. One of the selected clones secreting rhG-CSF, produced 100-120 mg/l of rhG-CSF three days post-induction with methanol. The recombinant cytokine was purified using two-step, ion-exchange chromatography. The final yield of purified G-CSF was 35 mg/L of culture medium. The biological activity of rhG-CSF was examined in rat bone marrow cells. The P. pastoris strain was designed to produce relatively high levels of rhG-CSF. The rhG-CSF protein had a strong stimulating effect on the growth of rat bone marrow cells, which was comparable to that of the commercial drug leukostim, but showed a more persistent effect on granulocyte cells and monocyte sprouts, enabling the enhanced maintenance of the viability of the cells into the 4th day of incubation.

[Effect of a gel based on recombinant human angiogenin on the healing of donor palate wounds]. / Vliianie gelia na osnove rekombinantnogo angiogenina cheloveka na zazhivlenie donorskikh ran tverdogo neba.

The aim of the study was to study the effect of the gel on the basis of recombinant human angiogenin on the rate of regeneration of donor palatal wounds. The study involved 20 patients (8 men and 12 women) aged 32 to 55 years. Patients were divided into two groups: the 1st group is a study group (n=10), whose patients in the postoperative period used a gel based on recombinant human angiogenin, the 2nd group is a control group (n=10) in which a gel based on recombinant human angiogenin was not used. Patients in both study groups underwent vestibuloplasty with simultaneous plasty of the attached keratinized gingiva with a free gingival graft from the area of the hard palate. The operations were carried out at the stage of disclosing dental implants, simultaneously with the installation of healing abatements or 4 weeks before dental implantation. For histological examination, tissue samples were obtained from the region of the edge of the donor's wounds of the palate at the 7th and 14th days after surgery. As a result of the study, significant differences were found in the comparison groups when assessing the processes of inflammation, angiogenesis and epithelization. The local application of the gel containing recombinant human angiogenin resulted in a rapid decrease in the intensity of inflammation in lamina propria mucosae and a significant decrease in the bulk density of cell infiltrates, accelerating regeneration. This is primarily due to the stimulation of the development of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and increased blood supply to the affected area, as well as an increase in the proportion of fibroblasts. The most important observation was the increase in the rate of epithelialization of donor wounds of the hard palate.

[IMMUNOCHEMICAL ANALYSIS OF RECOMBINANT CHIMERIC POLYPEPTIDE OspC(gar+afz) OF BORRELIA GARINIIANIP B. AFZELI ISOLATES].

AIM: Comparative study of antigenic properties of recombinant proteins OsPCgar and OsPCafz and recombinant chimeric polypeptide OspCgar+afrz, that contains amino acid sequences of mature immune dominant OspC proteins of West-Siberian isolates of Borrelia garinii (OspCgar) and B. afzelii (OspCafz), and evaluation of possibility of their use as antigens during creation of test-systems for serodiagnostics of Lyme borreliosis (LB) on the territory of Western Siberia. MATERIALS AND METHODS: Recombinant chimeric polypeptide OSpCgar+af, and recombinant mature proteins OSPCgar and OspCafz, obtained by expression of the corresponding genes in Escherichia coli cells; purified by affinity chromatography in Ni-NTA-sepharose CL-6B and studied by EIA method for the ability to bind antibodies from sera of LB patients. RESULTS: A difference in sensitiv- ity of determination by EIA method of specific IgM and IgG against borreliae in blood sera of LB patients with localized stage of the disease during use of OspCgar,'OSPCafz and OspCgar+afz chimera as antigens was shown. Chimeric antigen OSPCgar+afz was established to show higher antigenic activity compared with each of the OspCgar or OSPCafz antigens separately. CONCLUSION: The results of the study allow to examine.the recombinant chimeric polypeptide OspCgar+afz as a pos- sible component during creation of test-systems for serodiagnostics of LB on the territory of West, Siberia.

Distribution and species composition of potato viruses in the Novosibirsk region.

Among the many diseases that affect potato plants, viral infections are the most common and cause significant damage to farms, affecting both the yield and quality of potatoes. In this regard, an important condition for preserving the potato seed fund in Russia is systematic monitoring and early highly specific detection of potato viral infections. The purpose of the work is to study samples of potato varieties collected in the Novosibirsk region for the presence of viral infections using RT-PCR. 130 potato plants from three districts of the Novosibirsk region (NR) were studied. As a result of monitoring, the following viruses were identified: PVY (potato virus Y), PVS (potato virus S), PVM (potato virus M) and PVX (potato virus X). The quarantine pathogen potato spindle tuber viroid (PSTVd) was not detected in any of the samples analyzed. The maximum frequency of occurrence in the region was noted for three viruses: PVY, PVM and PVS. A significant proportion of the samples were mixed viral infections: the occurrence of the combination of infection PVY + PVM in plants was 25.0 %, and PVY + PVS, 22.6 %. To develop methods for determining the strain affiliation of the studied samples, the nucleotide sequences of the capsid protein genes of 10 Y-virus isolates were sequenced. Phylogenetic analysis of the studied sequences of NR isolates was carried out with a set of sequences of reference strains 261-4, Eu-N, N:O, NE-11, NTNa, NTNb, N-Wi, O, O5, SYR_I, SYR_II and SYR_III retrieved from GenBank. As a result of phylogenetic analysis, it was established that NR viral samples fell into two groups of strains: group 1, which also includes isolates of the reference strains 261-4/SYR_III, and group 2, NTNa. The obtained results of the strain affiliation of NR samples lay the basis for the development of DNA and immunodiagnostic systems for identifying PVY circulating in NR, as well as for elucidating the source and routes of entry of specific virus strains.Key words: Solanum tuberosum; viral infections; RT-PCR; potato Y virus; phylogenetic analysis.

[Evaluation of significance of Borrelia garinii spirochete recombinant protein DbpB for serodiagnostics of ixodes tick-borne borreliosis].

AIM: Obtaining recombinant protein DbpB of West Siberia Borrelia garinii 20047 isolate and evaluation of its antigen activity for the possible use in serodiagnostics of ixodes tick-borne borreliosis (ITB). MATERIALS AND METHODS: Coding region of dbpB gene of novosibirsk B. garinii 20047 isolate was amplified by PCR and cloned as part of expressing pETm vector in Escherichia coli BL21 (DE3) strain cells. Recombinant protein DbpB produced by the selected clone was studied by EIA method for its ability to react with sera antibodies of ITB patients. RESULTS: E. coli BL21 (DE3) clone producing recombinant protein DbpB in quantity of 30% of total E. coli cell protein was obtained. Homology of amino acid sequence of recombinant protein DbpB of novosibirsk B. garinii 20047 isolate with primary structures of B. garinii, B. afzelii and B. burgdorferi sensu stricto spirochete genospieces DbpB proteins presented in GenBank database was 98.4, 77 and 73%, respectively. Sensitivity of immune enzyme detection in sera of ITB patients with migrating erythema of IgM and IgG reacting with DbpB antigen was 13.9 and 20.0%, respectively. Frequency of detection of IgM and IgG against DbpB in patient sera with disseminated ITB form was 15.7 and 43.8%, respectively. Specificity of immune enzyme detection of antibodies against recombinant antigen DbpB in which sera of syphilis, rheumatoid arthritis patients and healthy donors used as control sera was 100%. CONCLUSION: DbpB recombinant protein of novosibirsk B. garinii 20047 isolate may be used as one of antigens for highly specific serodiagnostics of ITB disseminated stage.

[Recombinant strain producing thermostable lipase from Thermomyces lanuginosus immobilized into nanocarbon silica matrices and properties of the prepared biocatalyzers].

Multicomponent composite biocatalyzers with lipolytic activity have been studied. These biocatalyzers were prepared through the immobilization of a recombinant producer strain of thermostable lipase from Thermomyces lanuginosus into SiO2 xerogel, which contains a nanocarbon component, i.e., multilayered carbon nanotubes with varying diameters, and also bulblike structured carbon nanospheres ("nanobulb"). The properties of lipase were studied both in cell suspensions of a recombinant producer strain constructed based on E. coli BL21(DE3) and in the immobilized state with regard to the structure and dispersibility of the nanocarbon component used in the composition of the biocatalyzers. It was shown that the recombinant intracellular lipase exerted its activity in a reaction of tributirin hydrolysis on average comprising 50 U/mg of dried cells and had a high level of thermostability. Upon heating in olive oil at 100 degrees C, the inactivation constant and the period of semi-inactivation comprised 6 x 10(-3) min(-1) and 2 h, respectively, exceeding by one order the thermostability of lipase in a buffer solution. Biocatalyzers that contained aggregated "thick" nanotubes with a diameter of 20-22 nm had the maximum initial activity-250 U/g.

[Immunochemical analysis of recombinant protein FlaA of Western Siberian Borrelia garinii isolate].

AIM: Study of the ability of Western Siberian Borrelia garinii 20047 isolate recombinant protein FlaA to react with sera antibodies of ixodes tick borreliosis patients. MATERIALS AND METHODS: Recombinant antigen FlaA, sera of ixodes tick borreliosis patients, genetic engineering methods, solid phase EIA, and parametric and nonparametric statistical methods were used in the study. RESULTS: Recombinant form of mature flagellar protein FlaA of B. garinii 20047 was obtained. In EIA study of sera of ixodes tick borreliosis patients with migrating erythema and without it, IgM or IgG against FlaA antigen were detected in more than 30% of sera. Indicator of the detection of IgM against FlaA antigen in sera of ixodes tick borreliosis patients with migrating erythema and without it was 43.3% and 33.3% respectively. CONCLUSION: The results obtained show a significant antigenic activity of recombinant protein FlaA of Western Siberian B. garinii isolate and the perspectives of its use for serodiagnostics of ixodes tick borreliosis.

Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album.

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that - at a subsequent stage of transfection into yeast cells - it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein's secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 µg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

[Enzyme immunoassay-based analysis of OspC recombinant proteins from Borrelia garinii and Borrelia afzelii isolated in West Siberia].

AIM: To study the ability of OspC recombinant proteins from Borrelia garinii and Borrelia afzelii isolated in West Siberia to interact with serum antibodies from patients with tick-borne borreliosis (TBB). MATERIALS AND METHODS: Recombinant antigens OspC B. garinii and OspC B. afrelii, serum samples from patients with TBB were used as well as solid-phase enzyme immunoassay and parametric and non-parametric statistical methods. RESULTS: Higher antigenic activity of B. garinii OspC compared with OspC from B. afzelii was observed when these recombinant proteins were compared in enzyme immunoassay. Detection rate of class M and G immunoglobulins to B. garinii OspC in sera of patients with TBB was 60.5% and 70% respectively. CONCLUSION: Obtained results indicate high immunoreactivity of OspC recombinant proteins from B. garinii and B. afzelii and point to perspective of their combined use for serological diagnostics of TBB.

[Isolation of the recombinant proteins OspC and the fragment FlaA (F-FLAA) from the western-Siberian Borrelia garinii NT29 isolates and the study of their immunochemical properties].

The structural proteins OspC and FlaA of the Lyme disease (LD) agent are known to be the basic antigens, which induce the humoral immune response at the initial stage of the disease. The goal of this work was to obtain the recombinant OspC and a fragment of the FlaA protein (f-FlaA) from the Western Siberian Borrelia garinii NT29 isolates and to assess the possibility of their use for the LD diagnosis. Encoding regions of the OspC and f-FlaB genes were amplified using PCR inserted in the pREB expressive vectors and cloned in the E. coli str. BL21 and C-600, respectively. The recombinant OspC and f-FlaA proteins were purified using affinity chromatography on Ni-NTA-sepharose 6A, and their ability to bind serum antibody of patients with Lyme disease was tested using western-blot and ELISA methods. The results of the analyses suggest that these proteins can be considered as promising components for elaboration of diagnostic tests for LD. The prototype of the ELISA diagnostic test was designed on the basis of the OspC and f-FlaA recombinant antigens. This test provides satisfactory parameters of diagnostic specificity (70.0%) and sensitivity (85.0%).

[The 2003 results of monitoring of influenza A virus in the populations of wild birds in the south of Western Siberia].

The paper presents the results of isolation of influenza A virus from 97 cloacal swabs of 11 species of aquatic and semiaquatic wild birds collected on the Chany Lake (the south of Western Siberia, Ob-Irtysh interarea). Six strains with subtypes H2 (2 strains), H3 (3 strains), and H5 (1 strain) were isolated from mallard ducks (Anas platyrhynchos). The total infection rate in the examined birds was 6.2% and that in the ducks was 9.7%. The paper deals with the phylogenetic analysis of hemagglutinin of genes of isolates and with the comparison of the obtained results with the 2002 data in the same region. Analysis of H5 strain hemagglutinin proteolytic site permits one to regard this strain as non-pathogenic.

[Genetic variety of influenza A virus in the populations of wild birds in the south of Western Siberia].

Influenza A virus variants belonging to H3 and H4 subtypes were isolated from wild ducks inhabiting in the south of Western Siberia. Phylogenetic analysis of hemagglutinin (HA) gene of these viruses has revealed that H3 isolates are closely related to those isolated from the bird inhabiting in West Europe (A/Teal/Germany/wv01r/01, A/Duck/Ukraine/1/63) and China (A/Aquatic bird/Hong Kong/399/99); and those isolated from the birds inhabiting in Germany (A/Garganey/Germany/wv157k/01, A/Teal/Germany/wv153k/01). Thus, closely related influenza A virus variants circulate in the populations of the wild birds inhabiting in greatly spaced regions of Eurasia.

[Synthesis, cloning and primary structure of DNA complementary to mRNA for human pituitary pro-opiomelanocortin]. / Sintez, klonirovanie i opredelenie pervichnoi struktury DNK, komplementarnoi mRNK proopiomelanokortina iz gipofiza cheloveka.

cDNA coding for the human pro-opiomelanocortin (POMC) has been cloned and sequenced. It codes for full size amino acid sequence of POMC and furthermore, contains most part of the 3'-terminal noncoding mRNA region and 60 nucleotides coding for signal peptide.

[Synthesis, cloning and determination of the primary structure of a full-size DNA copy of the neuraminidase gene from influenza virus type A subtype H1N1]. / Sintez, klonirovanie i opredelenie pervichnoi struktury polnorazmernoi DNK-kopii gena neiraminidazy virusa grippa A podtipa H1N1.

Complete nucleotide sequence of the cloned full-length DNA copy of the influenza virus A (H1N1) neuraminidase gene has been determined. The predicted amino acid sequence is compared with sequences of neuraminidases from other influenza virus strains. A section of the neuraminidase is found to be homologous to the chicken lysozyme catalytic centre.

[Synthesis, cloning and determination of the primary structure of a full-size DNA copy of the NP protein gene from influenza virus type A]. / Sintez, klonirovanie i opredelenie pervichnoi struktury polnorazmernoi DNK-kopii gena belka NP virusa grippa A.

Complete nucleotide sequence of the cloned full-length DNA copy of the influenza virus A (H1N1) RNA segment 5 has been determined. The degree of nucleotide difference between two variants of A/PR/8/34 strain is estimated. The possible secondary structure of the segment 5 is deduced from the nucleotide sequence of some clones.

[Synthesis, cloning and determination of the primary structure of a full-size DNA copy of fragment 8 from the influenza virus type A genome]. / Sintez, klonirovanie i opredelenie pervichnoi struktury polnorazmernoi DNK-kopii fragmenta 8 genoma virusa grippa A.

Complete nucleotide sequence of the cloned full-length DNA copy of the influenza virus A (H1N1) RNA segment 8 has been determined. A section of the hypothetical protein coded for by the negative strand of the segment 8 is found to be homologous to the trypsin catalytic centre.

[Primary structure of the full-size DNA copy of the NP gene of influenza virus A/Kiev/59/79 (H1N1)]. / Pervichnaia struktura polnorazmernoi DNK-kopii gena belka NP virusa grippa A/Kiev/59/79 (H1N1).

The complete nucleotide sequence of the cloned full-length DNA copy of A/Kiev/59/79 (H1N1) influenza virus nucleoprotein gene has been determined. This strain is shown to be the natural recombinant that inherited its nucleoprotein gene from contemporary H3N2-influenza strains. The comparison with other NP-genes reveals the probable localization of antigenic determinants and phosphorylation site of the NP-protein.

[Primary structure of the full-size DNA copy of the hemagglutinin gene of influenza virus A/Kiev/59/79 (H1N1)]. / Pervichnaia struktura polnorazmernoi DNK-kopii gena gemaggliutinina virusa grippa A/Kiev/59/79 (H1N1).

The complete nucleotide sequence of the cloned full-length DNA copy of A/Kiev/59/79 (H1N1) influenza virus hemagglutinin gene has been determined. The comparison with the other hemagglutinin structures reveals the divarication of evolutionary pathway of the H1N1-influenza viruses.

[Synthesis of a full-length DNA copy of the hemagglutinin gene of the the influenza virus A H1N1 subtype, its cloning and primary structure]. / Sintez polnorazmernoi DNK-kopii gena gemaggliutinina virusa grippa A H1N1-podtipa, ee klonirovanie i opredelenie pervichnoi struktury.

Full-length DNA-copy of hemagglutinin gene RNA of influenza A virus strain A/Leningrad/54/1 was synthesized and cloned in E. coli plasmid pBR 327. Its primary structure was determined by a modified Maxam - Gilbert procedure. The nucleotide sequence of this gene was compared with sequences of analogous genes of influenza strains A/USSR/90/77 and A/PR/8/34. An addition of one nucleotide in non-translated region was found in the former.