Development of shortened recombinant flagellin and study of its radioprotective efficacy in mice with acute radiation injury

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Abstract

The aim of the work is an experimental study of the radioprotective effect of shortened recombinant flagellin under conditions of total body irradiation of mice by survival rates and the effect on hemo- and immunopoiesis. A new recombinant shortened derivative of Salmonella enterica – dFliC protein obtained by structure-oriented reengineering of a previously developed molecule was used. The radioprotective effect of dFliC was studied in the mouse model of hematopoietic acute radiation syndrome. The 30-day survival of irradiated mice was analyzed using the Kaplan-Meier method. The effect of dFliC protein on the number of splenic colony-forming units (CFU-s) and myelokaryocytes in the bone marrow, peripheral blood parameters, and the cytokine profile in the blood serum of mice was assessed. Statistica 8.0 software was used for statistical processing of the results. A purification test record for the recombinant flagellin dFliC was developed, and protein samples with a purity of 92.79% according to HPLC were obtained. Administration of dFliC at a dose of 1 mg/kg 30 min prior to 7.8 Gy X-rays increased 30-day survival of mice by 38% (p < 0.05 compared to the vehicle control). On day 9 after X-ray irradiation at a dose of 7 Gy, the number of colonies increased 2.8 times in dFliC-treated mice (p < 0.05), the viability of hematopoietic progenitor cells increased and the severity of thrombocytopenia decreased. An increase in the production of cytokines involved in hematopoiesis, IL-3. GM-CSF and IL-12p40, has been recorded. The level of proinflammatory cytokines IL-1β and IL-33 was maintained at a lower level than when exposed to radiation without dFliC. Deimmunization and structural rearrangement of the flagellin molecule did not lead to a decrease in the radioprotective effect of the recombinant protein. Radioprotective efficacy of dFliC is provided by a protective effect on bone marrow hematopoietic cells and stimulation of postradiation restoration of hemo- and immunopoiesis by regulating the expression of cytokines with a wide range of biological activity.

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About the authors

Elena V. Murzina

Kirov Military Medical Academy

Author for correspondence.
Email: elenmurzina@mail.ru
ORCID iD: 0000-0001-7052-3665
Russian Federation, Saint Petersburg

Genrikh A. Sofronov

Kirov Military Medical Academy

Email: gasofronov@mail.ru
ORCID iD: 0000-0002-8587-1328
Russian Federation, Saint Petersburg

Andrei S. Simbirtsev

St. Petersburg Pasteur Research Institute of Epidemiology and Microbiology

Email: simbas@mail.ru
ORCID iD: 0000-0002-8228-4240
Russian Federation, Saint Petersburg

Natalia V. Aksenova

Kirov Military Medical Academy

Email: nataaks@mail.ru
ORCID iD: 0000-0002-5645-7072
Russian Federation, Saint Petersburg

Nicolay A. Klimov

Institute of Experimental Medicine, Saint Petersburg, Russia

Email: nklimov@mail.ru
ORCID iD: 0000-0002-5243-8085
Russian Federation, Saint Petersburg

Olga M. Veselova

Kirov Military Medical Academy

Email: veselova28@mail.ru
ORCID iD: 0009-0007-9345-1845
Russian Federation, Saint Petersburg

Elena V. Dmitrieva

Kirov Military Medical Academy

Email: ev.dmitrieva@yandex.ru
ORCID iD: 0000-0001-6514-7837
Russian Federation, Saint Petersburg

Vladimir V. Kopat

“ATG Service Gene” LLC

Email: kopat@service-gene.ru
ORCID iD: 0000-0002-6573-6743
Russian Federation, Saint Petersburg

Anastasia A. Riabchenkova

“ATG Service Gene” LLC

Email: riabchenkova@service-gene.ru
ORCID iD: 0000-0002-9973-0753
Russian Federation, Saint Petersburg

Evgenii L. Chirak

“ATG Service Gene” LLC

Email: chirak.evgenii@service-gene.ru
ORCID iD: 0000-0001-9167-5000
Russian Federation, Saint Petersburg

Elizaveta R. Chirak

“ATG Service Gene” LLC

Email: chirak.elizaveta@service-gene.ru
ORCID iD: 0000-0002-1610-8935
Russian Federation, Saint Petersburg

Ilya V. Dukhovlinov

“ATG Service Gene” LLC

Email: atg@service-gene.ru
ORCID iD: 0000-0002-5268-9802
Russian Federation, Saint Petersburg

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Supplementary files

Supplementary Files
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2. Fig. 1. Structure of the recombinant protein dFliC: A –3D structure of FliC UniProt ID P06179 domains, obtained using iTASSER, B – structure of FliC domains, C – structure of dFliC domains.

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3. Fig. 2. Electropherogram of the results of chromatographic purification of dFliC using hydrophobic chromatography (HIC) in 12% PAAG. M – Mixture of marker fragments PageRuler Protein Ladder Plus; 1 – dFliC, fraction applied to the column (8 μg); 2 – dFliC, unbound fraction from the column during HIC (10 μl); 3 – dFliC, target fraction obtained during HIC (10 μg).

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4. Fig. 3. HPLC analysis of a sample of purified recombinant dFliC protein using Agilent 1100 chromatograph using an analytical column Jupiter C18, 5 μm, 46,300 A°, 4.6 × 250 mm (Phenomenex, USA). Retention time: 14.294 min. Purity of target protein: 92.79%.

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5. Fig. 4. Survival curves of mice after total body X-ray irradiation at a dose of 7.8 Gy without treatment and the use of flagellincontaining proteins (“рФЛ” and “dFliC”) at a dose of 1 mg/kg or recombinant interleukin-1β (“ИЛ-1β”), 50 mg/kg.

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6. Fig. 5. The number of endogenous colony-forming units on the spleens of mice on day 9 after total body X-ray irradiation and the use of dFliС at a dose of 1 mg/kg.

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7. Fig. 6. Number of myelokaryocytes in the mouse bone marrow on day 9 after total body X-ray irradiation without treatment or using dFliС. * р < 0.005; ** – р < 0.01 vs unirradiated mice (Mann–Whitney U Test)

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8. Fig. 7. Platelet count in the blood of mice on day 9 after total body X-ray irradiation at doses of 7 and 7.8 Gy without treatment or using deimmunized recombinant flagellin (dFliС). * р < 0.05; ** р < 0.005 vs unirradiated mice (Mann–Whitney U Test).

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9. Fig. 8. Cytokine concentration (pg/ml) in the blood serum of mice on day 9 after total body X-ray irradiation at a dose of 7 and 7.8 Gy alone or using deimmunized recombinant flagellin (dFliС) at a dose of 1 mg/kg. * Differences are statistically significant with the “Kbiol” group, p < 0.05 (Mann–Whitney test).

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