biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2026-746biopreparat-746Research ArticleРЕКОМБИНАНТНЫЕ БЕЛКИRECOMBINANT PROTEINSБифункциональные гибридные белки Z-mHoneydew и Z-CyOFP1 на основе Z-домена белка A Staphylococcus aureus для иммунофлуоресцентного анализа иммуноглобулинов G в сыворотке кровиBifunctional hybrid proteins Z-mHoneydew and Z-CyOFP1 based on the Z-domain of Staphylococcus aureus protein A for immunofluorescence assay of immunoglobulins G in blood serumhttps://orcid.org/0000-0002-9134-5586ЕлисейкинА. М.EliseykinA. M.

Eлисейкин Алексей Михайлович

пр. Ленинградский, д. 10, Кемерово, 650065

Alexey M. Eliseykin

10 Leningradsky Ave., Kemerovo 650065

aleksej.elisejkin.98@bk.ruhttps://orcid.org/0009-0001-7602-0230МаргатскийИ. В.MargatskiyI. V.

Маргатский Иван Владимирович 

пр. Ленинградский, д. 10, Кемерово, 650065

Ivan V. Margatskiy

10 Leningradsky Ave., Kemerovo 650065

АртемовВ. Е.ArtemovV. E.

Артемов Владислав Евгеньевич

пр. Ленинградский, д. 10, Кемерово, 650065

Vladislav E. Artemov

10 Leningradsky Ave., Kemerovo 650065

https://orcid.org/0000-0002-9560-7563МарущакА. В.MarushchakA. V.

Марущак Анна Владимировна 

пр. Ленинградский, д. 10, Кемерово, 650065

Anna V. Marushchak

10 Leningradsky Ave., Kemerovo 650065

ПонкратенкоИ. С.PonkratenkoI. S.

Понкратенко Игорь Сергеевич 

пр. Ленинградский, д. 10, Кемерово, 650065

Igor S. Ponkratenko

10 Leningradsky Ave., Kemerovo 650065

https://orcid.org/0000-0002-8560-6719ГлушковА. Н.GlushkovA. N.

Глушков Андрей Николаевич, д-р мед. наук 

пр. Ленинградский, д. 10, Кемерово, 650065

Andrey N. Glushkov, Dr. Sci. (Med.)

10 Leningradsky Ave., Kemerovo 650065

https://orcid.org/0000-0002-6623-8818СтуденниковА. Е.StudennikovA. E.

Студенников Артем Евгеньевич, канд. биол. наук 

пр. Ленинградский, д. 10, Кемерово, 650065

Artem E. Studennikov, Cand. Sci. (Biol.)

10 Leningradsky Ave., Kemerovo 650065

studennikovav@ficuuh.ruФедеральное государственное бюджетное научное учреждение «Федеральный исследовательский центр угля и углехимии Сибирского отделения Российской академии наук», Институт экологии человекаРоссияFederal State Scientific Institute, Federal Research Centre for Coal and Coal Chemistry, Siberian Branch of the Russian Academy of Sciences, Institute of Human EcologyRussian Federation202604072026262196207Copyright © Елисейкин А.М., Маргатский И.В., Артемов В.Е., Марущак А.В., Понкратенко И.С., Глушков А.Н., Студенников А.Е., 20262026Елисейкин А.М., Маргатский И.В., Артемов В.Е., Марущак А.В., Понкратенко И.С., Глушков А.Н., Студенников А.Е.Eliseykin A.M., Margatskiy I.V., Artemov V.E., Marushchak A.V., Ponkratenko I.S., Glushkov A.N., Studennikov A.E.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.biopreparations.ru/jour/article/view/746ВВЕДЕНИЕ

ВВЕДЕНИЕ. Традиционные методы обнаружения иммуноглобулинов G (IgG) в сыворотке крови основаны на применении антител, химически конъюгированных с ферментными или флуоресцентными метками. Эффективность таких методов ограничивается стерическими помехами, возникающими в процессе химической конъюгации. В качестве альтернативы для иммунофлуоресцентного анализа IgG предложены бифункциональные гибридные белки Z-mHoneydew и Z-CyOFP1 на основе Z-домена белка A Staphylococcus aureus и ярких флуоресцентных белков mHoneydew и CyOFP1, которые сочетают способность связываться с IgG и интенсивную флуоресценцию, а их получение исключает этап химической конъюгации.

ЦЕЛЬ

ЦЕЛЬ. Получение гибридных белков на основе Z-домена белка A для создания универсальных флуоресцентных реагентов для обнаружения IgG в сыворотке крови.

МАТЕРИАЛЫ И МЕТОДЫ

МАТЕРИАЛЫ И МЕТОДЫ. Генетические конструкции pCCO-Z-mHoneydew и pCCO-Z-CyOFP1 получали методами молекулярного клонирования в вектор pCCO. Экспрессию белков Z-mHoneydew и Z-CyOFP1 (~35 кДа) проводили в Escherichia coli штамм M15. Белки очищали аффинной хроматографией на Ni²⁺-NTA агарозе. Свойства белков изучали с использованием спектрофлуориметрии, иммуноферментного анализа (ИФА) и иммунофлуоресцентного анализа.

РЕЗУЛЬТАТЫ

РЕЗУЛЬТАТЫ. Генетические конструкции pCCO-Z-mHoneydew и pCCO-Z-CyOFP1 под контролем промотора T5 кодируют гибридные белки со следующей структурой: Z-домен белка A — гибкий линкер (Ser-Ser-Ser-Gly-Ser-Ser-Ser-Gly) — флуоресцентный белок mHoneydew или CyOFP1 — гексагистидиновая метка. При экспрессии выход растворимых белков составил ~37 мг/л (Z-mHoneydew) и ~6 мг/л (Z-CyOFP1) с чистотой ≥90%. Максимумы возбуждения и эмиссии Z-mHoneydew зарегистрированы при длинах волн 480 и 536 нм соответственно со сдвигом эмиссии в синюю область на 26 нм относительно нативного mHoneydew (562 нм). Максимум возбуждения Z-CyOFP1 зарегистрирован при 515 нм со сдвигом в красную область на 18 нм относительно нативного CyOFP1 (497 нм) при сохранении максимума эмиссии. Методом ИФА показано дозозависимое связывание IgG c гибридными белками. Для Z-mHoneydew линейная зависимость наблюдалась в диапазоне концентраций от 30 до 235 нг/мкл (R²=0,96), а для Z-CyOFP1 — от 15 до 250 нг/мкл (R²=0,97), что подтверждает способность гибридных белков связываться с IgG. Данные иммунофлуоресцентного анализа указали на возможность обнаружения IgG, специфических к конъюгату бензо[а]пирена с бычьим сывороточным альбумином, в сыворотке крови кролика с использованием гибридных белков. Линейная зависимость для Z-mHoneydew наблюдалась в диапазоне разведений сыворотки 1:10–1:160 (R²=0,92), а для Z-CyOFP1 — 1:10–1:80 (R²=0,98), что свидетельствует о пригодности данных белков для обнаружения специфических антител в сыворотке крови.

ВЫВОДЫ

ВЫВОДЫ. Гибридные белки Z-mHoneydew и Z-CyOFP1 сохраняют флуоресцентные свойства и способность связываться с IgG, в том числе с IgG сыворотки крови, что перспективно с точки зрения разработки прототипов универсальных иммунофлуоресцентных тест-систем.

INTRODUCTION

INTRODUCTION. Traditional methods for detecting serum IgG rely on antibodies chemically conjugated to enzymatic or fluorescent tags. The efficiency of such methods may be limited by steric hindrance during chemical cross-linking. As an alternative, bifunctional hybrid proteins Z-mHoneydew and Z-CyOFP1, based on the Z-domain of Staphylococcus aureus protein A and bright fluorescent proteins mHoneydew and CyOFP1, combining the binding to IgG and intense fluorescence, have been proposed for IgG immunofluorescence analysis; their production excludes the stage of chemical conjugation.

AIM

AIM. This study aimed to produce hybrid proteins based on the Z-domain of protein A for the creation of universal fluorescent reagents for the detection of IgG in blood serum.

MATERIALS AND METHODS

MATERIALS AND METHODS. The pCCO-Z-mHoneydew and pCCO-Z-CyOFP1 genetic constructs were created by molecular cloning in the pCCO vector. Z-mHoneydew and Z-CyOFP1 proteins (~35 kDa) were expressed in Escherichia coli strain M15. Protein purification was performed by affinity chromatography on Ni²⁺-NTA agarose. The protein properties were studied using spectrofluorimetry, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay.

RESULTS

RESULTS. Genetic constructs pCCO-Z-mHoneydew and pCCO-Z-CyOFP1 under the control of the T5 promoter encode chimeric proteins with the following structure: Z-domain of protein A — flexible linker (Ser-Ser-Ser-Gly-Ser-Ser-Ser-Gly) — fluorescent protein mHoneydew or CyOFP1 — 6×His tag. Soluble proteins were obtained with a yield of ~37 mg/L (Z-mHoneydew) and ~6 mg/L (Z-CyOFP1) with a purity ≥90%. For the Z-mHoneydew protein, excitation and emission maxima were observed at 480 and 536 nm, respectively; a 26 nm blue shift in emission was observed relative to native mHoneydew (562 nm). For Z-CyOFP1, the excitation maximum was recorded at 515 nm; an 18 nm red shift in excitation was observed relative to native CyOFP1 (497 nm), while maintaining the emission maximum. Dose-dependent binding of IgG to the hybrid proteins was demonstrated using ELISA. A linear dependence was observed for Z-mHoneydew in the concentration range of 30–235 ng/μL (R²=0.96) and 15–250 ng/μL (R²=0.97) for Z-CyOFP1, confirming the capacity of the hybrid proteins to bind IgG. Immunofluorescence assay showed the feasibility of detecting IgG, specific to conjugate benzo[a]pyrene-bovine serum albumin, in rabbit serum using hybrid proteins. A linear relationship was observed for Z-mHoneydew in the serum dilution range of 1:10–1:160 (R²=0.92) and 1:10–1:80 (R²=0.98) for Z-CyOFP1, evidencing the suitability of hybrid proteins for detection of specific antibodies in blood serum.

CONCLUSIONS

CONCLUSIONS. Hybrid proteins Z-mHoneydew and Z-CyOFP1 retain fluorescent properties and the capacity to bind to IgG, including serum IgG, which is promising from the point of view of developing prototypes of universal immunofluorescent test systems.

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The authors declare that there are no conflicts of interest present.