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Registration Number

0215U003391, 0110U005967 , R & D reports

Title

RECOMBINANT BLOOD COAGULATION SYSTEM PROTEINS GENERATION AND THEIR CHARACTERISTICS INVESTIGATION FOR DIC-SYNDROME DIAGNOSTICS AND TREATMENT.

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Head

Komisarenko Serhiy, Доктор біологічних наук

Registration Date

28-01-2015

Organization

A.V.Palladin Institute of Biochemistry of National Academy of Sciences

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The aim of this project was the generation of monoclonal antibodies and recombinant scFv-antibodies against human protein C. These antibodies should be suitable for generation of immunochemical methods for human plasma protein C detection in case of disseminated intravascular coagulation syndrome (DIC-syndrome) and trombofilia. Also during this work stable cell lines for recombinant protein C generation should be obtained. This protein C could be used for DIC-syndrom therapy. In this study modern chemical, biochemical, bioinformatical, immunochemical and molecular biology methods were used. During the project highly immunogenic unique aminoacid sequences of protein C molecule were determined, appropriate peptide synthesized and with carrier proteins conjugated. Splenocytes of mice immunized with KLH-Pro144-Leu155 were hybridized with myeloma Х-63Ag8 cells.Stable clone producer of monoclonal antibodies against Pro144-Leu155 fragment of human ptotein C was obtained. It was shown that monoclonal antibodies ІV-6А which have been produced by this clone specific bind human protein C and could be used for design immunoenzymes test-systhems for protein C detection in blood plasma. Also it was shown that monoclonal antibodies ІV-6А could be used for protein C elution and purification by affinity chromatography. Immune libraries of immunized with protein C and Pro144-Leu155 protein C region-KLH conjugate mice genes were generated. The library was screened against Pro144-Leu155 protein C region-KLH and -BSA conjugates and one scFv producer clone was isolated. Recombinant anti-protein C scFv producing clones were selected. The best clone DNA sequence was subcloned into pET-22b expression vector and recombinant anti-protein C scFv producer based on E. coli Rosetta was obtained. It was shown that obtained 9Е scFv antibodies could specific bind protein C in immunoensyme analysis and immunoblotting and could be used for design of immunochemical methods for protein C detection. Two genetic construction for protein C expression in eukaryotic cells based on pEGFP plasmid were design. They contain CMV or EF-1alfa promoter. 3Т3, HEK293 and CHO cell lines were transfected with these plasmids. Then protein C expression in these cells were investigated. One effective recombinant protein C producing clone was selected from transected with pEGFP-N1-protein C HEK293 cells. Recombinant scFv antibodies 9E and monoclonal antibodies ІV-6А against protein C would be used for design of immunochemical test systhems for protein C detection in human blood plasma, which is important for trombofilia and DIC-syndrome diagnostics as well as affinic sorbents for protein C elution and purification. Recombinant protein C producer could be used for obtaining of protein C as antithrombotic and aceptic agent for DIC-syndrome and other human deseases treatment.

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Богатирьова О. А.
Колеснікова І. М.
Костюченко О. П.
Литвинова Л. М.
Луговська Н. Е.
Луговськой Е. В.
Макогоненко Є. М.
Олійник О. С.
Паливода К. О.
Пидюра М.О.

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2020-04-02