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Senior investigator
The effects of intraparenchymal injection of recombinant cytokines on the morphofunctional state of the kidney in chronic experimental ischemia
The effects of intraparenchymal injection of recombinant cytokines on the morphofunctional state of the kidney in chronic experimental ischemia
The studies were performed in collaboration with SI “Institute of Urology named by academician O.F.Vozianov of NAMS of Ukraine” It has been demonstrated in the experimental model of chronic kidney ischemia in rabbits that the intraparenchymal administration of the FGF-2 in developed polymeric carrier, based on cross-linked heparin, 5 months after ligature application, decreases the initial sclerotic changes in stroma and vessels and protects from the development of irreversible sclerotic changes in the interstitium and atrophy of the elements of parenchyma in ischemic and contralateral kidney. The data of angiography indicate that the architectonics of arterial bed of kidney of experimental animals with segmental ischemia was almost the same as of intact contralateral kidney after the injection of the preparation of FGF-2 studied (Fig. 4). The gene coding for human interleukin-10 has been cloned, the high-level expression of this cytokine in E.coli cells has been obtained, and the method for its downstream purification and obtaining in biologically active form has been elaborated in the Department. It has been shown that the intraparenchymal administration of the interleukin-10 into ischemic kidney tissue reduced the activity of the processes of lipid peroxidation and decreased the risk of ischemic kidney damage. The genes coding for human stromal-derived factor-1α (SDF-1α) and vascular endothelial growth factor have been cloned for further studies. The expression of these recombinant proteins in E.coli cells has been obtained, and the method for downstream purification and obtaining in biologically active form of SDF-1α has been elaborated.
Angiogram of vessels of rabbit urinary system after 6 months the application of ligatures on the upper pole of left kidney. The place of the ligature application shown by arrow
Senior investigator
Production, Purification and Detection of Specific Polyclonal Antibodies
Production, Purification and Detection of Specific Polyclonal Antibodies
Рrotein A Staphylococcus aureus (SPA) is a cell wall associated protein exposed on the surface of the S. aureus. SPA has high affinity to IgG from various animal species and human. The most applied affinity system for the purification of antibodies is based on SPA. The DNA sequences encoding SPA and cellulose binding domain CBD were genetically fused, expressed in E. coli system in a soluble form. The SPA-CBD2 fusion protein was affinity-immobilized on the microcrystalline cellulose CC31 and was used for purification of antibodies. The purity of antibodies in eluted fractions was more than 95 %.. Introduced C-terminal cysteine provided oriented covalent immobilization of SPA on maleimide-activated matrix.
In addition to affinity chromatography, the use of SPA, fused with a tag (enzymes, fluorescent proteins) is promising for diagnostics. As a fusion partner for the SPA was selected BAPmut. SPA-BAPmut was expressed in a soluble form in E. coli system. The target protein was obtained with purity more than 95 % using metal affinity chromatography. SPA-BAPmut is thermostable, and both parts of fusion protein (SPA and BAPmut) retain their IgG binding and alkaline phosphatase activity for a long time. The possibility of using SPA-BAPmut as universal secondary immunoreagent for different types of immunoassays was shown.
A highly efficient and inexpensive laboratory method of production and purification of polyclonal antibodies against the human cell surface CD34 marker and recombinant human Interleukin 7 was developed. It was demonstrated that unglycosylated recombinant protein cloned in E. coli cells and containing the extracellular fragment of the human CD34 antigen or IL 7 maintained the necessary antigenic determinants during isolation from bacteria and during immunization, induced the production of specific polyclonal antibodies, which could recognize the native antigen on the cell surface. The obtained antibodies can be used for CD34+ cell phenotyping by the immunocytochemistry and flow cytometry methods. Antibodies against IL 7 can be used for immunology research.
Senior investigator
Recombinant Antibody Expression and Purification
Recombinant Antibody Expression and Purification
A single-chain fragment variable (scFv) consists of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short peptide linker. The molecular weight is approximately 25 kDa. scFv has smaller size compare to full length antibodies and can be easily fused with target molecule such us enzymes, fluorescent proteins etc. scFv has great potential for it’s application for as research, diagnostic and medical applications. The technology for production of Recombinant Antibody against therapeutically important antigens such us Interferon alfa-2b (Pavlo Gilchuk), Interferon beta-1b (Maryna Pavlova), cell surface marker CD34 (Iulian Nikolaiev) and its fusion with target proteins (Maryna Tsapenko, Andrew Flyak, Oksana Gorbatiuk) was developed in our department.
A laboratory method for obtaining immunoaffinity media for chromatographic purification of proteins based on orienting and non-covalent immobilization of recombinant antibody fragments on cellulose matrix was obtained. The ScFv against human IFN-a2b and IFN-beta-1b was genetically fused to cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome and expressed in Escherichia coli. After the iso la tion of the target protein in func tion ally ac tive form from bac te ria cells the di rected im mo bi li za tion on microgranular cel lu lose has been carried out. The cellulose with immobilized ScFv-CBD-fusion was used as affinity media to per form the purification of recombinant human IFN-a2b and IFN- beta-1b.
Senior investigator
Development of effective methods of obtaining recombinant fusion proteins rhIL7
Development of effective methods of obtaining recombinant fusion proteins rhIL7
Human interleukin-7 (hIL-7) is a therapeutically important immune cytokine that significantly affects the maturation and homeostasis of T and B lymphocytes. Numerous studies of IL-7 have shown that its level substantially varies in various diseases, such as viral infections (HIV, cytomegalovirus infection), multiple sclerosis, rheumatoid arthritis, and others. Obtaining anti-human IL-7 specific antibodies, which are used in modern fundamental and applied biological and medical diagnostic research, is promising. New approaches are being developed to increase the efficiency of antibody production and purification. To obtain monoclonal antibodies one can use affinity sorbents based on Staphylococcus aureus protein A and Streptococcus sp. protein G. In the case of obtaining polyclonal antibodies, this approach does not allow to obtain highly specific antibodies that can critically affect the results of enzyme-linked immunosorbent assays. The use of other chromatographic approaches based on the physicochemical properties of antibodies cannot provide the required degree of purity. In turn, purification of antibodies using an antigen immobilized on the sorbent through a partner protein or affinity tag does not require a chemical modification of the matrix and antigen. Therefore, effective methods of obtaining recombinant fusion proteins rhIL7 with oligo-histidine tag (rhIL7-His), rhIL7 with cellulose-binding domain (CBD) from the cellulosome of Clostridium thermocellum (rhIL7-CBD), and rhIL7 with bacterial alkaline phosphatase (rhIL7-BAPmut) were developed.
To verify rhIL7-His protein activity a comparative analysis with a standard rhIL7 was performed on peripheral blood mononuclear cells (PBMCs). The response of PBMCs to the rhIL7-His was measured in the MTT assay for cell viability using phytohemagglutinin-activated human peripheral blood lymphocytes.
The rhIL7-CBD fusion protein, after immobilization on microcrystalline cellulose CC31, and rhIL7-His, after immobilization on a metal-affinity sorbent, were used to obtain polyclonal antibodies with specificity for human interleukin 7, with a purity of more than 95 %. Obtained bifunctional fusion protein rhIL7-BAPmut was used for screening of immunoglobulin variable genes of immune combinatorial cDNA libraries.
Очищення IgG, із сироваток крові мишей, на біоафінному сорбенті з іммобілізованим білком А Staphylococcus aureus: а – хроматограма (1 – нанесення білків сироваток крові; 2 – білки, які видаляються при промиванні сорбенту; 3 – елюція очищених антитіл); б – електрофореграма фракцій білків (1 – білки сироватки крові, які наносили на сорбент; 2 – білки, які не зв’язалися із сорбентом; 3 – очищена фракція IgG; М – маркери молекулярної маси)Angiogram of vessels of rabbit urinary system after 6 months the application of ligatures on the upper pole of left kidney. The place of the ligature application shown by arrow
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