The main idea of the project is to develop an immunochromatographic assay based on the recombinant P32 antigen of the lumpy skin disease virus and monoclonal antibodies to this protein. The project is aimed at solving problems with the spread of the lumpy skin virus in farm animals in the Republic of Kazakhstan. The resulting express test system will reduce the time of diagnosis to 10-20 minutes. The test system is designed to detect antibodies in the serum of cattle suffering from lumpy skin disease.
Lumpy skin disease (LSD) is a transboundary disease and poses a serious threat to livestock production, having a great impact on the economic situation both within our country and in the world as a whole.The morbidity rate among unvaccinated cattle reaches 85%, and the mortality rate is not high, but in some cases reached 40%. Today, in accordance with the requirements of the International Office of Epizootics, cases of LSD are subject to mandatory notification. The causative agent of LSD is a virus belonging to the capripoxvirus family, which also includes Shippox and Goatpox viruses. The disease leads to serious losses in animal productivity, abortions and problems during subsequent insemination, infertility of breeding bulls, damage to the skin, a decrease in live body weight, and costs for health-improving measures. Transmission of the virus from sick animals to healthy ones occurs through blood-sucking insects.
In Kazakhstan, LSD was first registered in 2016, in the Atyrau region. Presumably, the virus was introduced from the territory of a neighboring country, the Russian Federation, where LSD was first registered in 2015. To prevent the disease, 370 thousand heads of cattle were vaccinated in 2018, which had a positive impact on the epizootic situation regarding lumpy skin disease in the country. Considering the widespread prevalence of the virus, the development of an immunochromatographic test based on recombinant antigen and monoclonal antibodies for the diagnosis of LSD in farm animals is relevant for the Republic of Kazakhstan and has scientific novelty.
The final product of the study will be an immunochromatographic test for the diagnosis of LSD. Currently, immunochromatographic test systems around the world are increasingly used in various industries. Immunochromatographic tests are used to determine infectious and internal, non-contagious diseases of humans and animals, to detect drugs and antibiotics in food, etc. It should be noted that in the Republic of Kazakhstan, the use of immunochromatographic test systems is mainly limited to the medical field in determining pregnancy.
Increased interest in immunochromatographic analysis is associated with its rapidity (diagnosis within 10-20 minutes), efficiency (does not require much labor or specialized equipment, which allows examination in the field), and fairly high specificity and sensitivity during mass examination. The availability of an immunochromatographic test for the serological diagnosis of LSD will allow farmers to protect themselves from purchasing sick livestock and thereby prevent further spread of the infection. Using this test system, veterinary specialists will be able to promptly identify sick animals and take the necessary measures in a timely manner.
Development of an immunochromatographic assay for the rapid diagnosis of LSD based on the recombinant P32 antigen of the virus and specific monoclonal antibodies.
As a result of the project, an immunochromatographic test will be developed for the diagnosis of LSD in cattle. Strains of E. coli microorganisms producing the recombinant P32 antigen of the LSD virus will be obtained. Diagnostic properties of the recombinant P32 antigen will be studied. Two hybridoma cell lines producing monoclonal antibodies against the recombinant P32 antigen will be obtained. The immunochemical and diagnostic properties of monoclonal antibodies against the P32 antigen will be studied.
Tursunov Kanat Akhmetovich, PhD, H-index 3 (Author ID Scopus – 57193579180, Researcher ID Web of Science N-6319-2017)
Adish Zhansaya Batyrbekkyzy, higher education, PhD student in biology. Researcher ID: AAW-7200-2020; Scopus Author ID: 57202535857; ORCID: 0000-0001-9527-8774.
Kanayev Darkhan Babanovich, higher education, master of biological sciences. Researcher ID: N-6950-2017; ORCID: 0000-0001-9569-9034; Scopus Author ID: 278641.
Tokhtarova Laura Abdikhalykovna, higher education, master of technological sciences. (ORCID:0000-0003-4386-993X).
1. Borovikov, S., Tursunov, K., Syzdykova, A., Begenova, A., Zhakhina, A. (2023) Expression of recombinant Omp18 and MOMP of Campylobacter jejuni and the determination of their suitability as antigens for serological diagnosis of campylobacteriosis in animals Veterinary World, 16(1), 222–228. https://doi.org/10.14202/vetworld.2023.222-228. Percentile – 80.
2. Tursunov, K., Tokhtarova, L., Kanayev, D., Mustafina, R., and Mukantayev, K. (2022) Effect of thioredoxin on the immunogenicity of the recombinant P32 protein of lumpy skin disease virus, Veterinary World, 15(10): 2384–2390. https://doi.org/10.14202/vetworld.2022.2384-2390. Q2. Percentile – 79.
3. Mukantayev, K., Kanayev, D., Zhumabekova, S., Shevtsov, A., Tursunov, K., Mukanov, K., and Ramankulov Y. (2022) Optimization of polymerase chain reaction for the identification of Roe deer, Saiga, and Siberian stag living in Kazakhstan, Veterinary World, 15(8): 2067–2071. https://doi.org/10.14202/vetworld.2022.2067-2071. Q2. Percentile – 79.
4. Adish Zhansaya, Mukantayev Kanatbek, Tursunov Kanat, Ingirbay Bakhytkali, Kanayev Darkhan, Kulyyassov Arman, Tarlykov Pavel, Mukanov Kasym, Ramankulov Yerlan. Recombinant Expression and Purification of Extracellular Domain of the Programmed Cell Death Protein Receptor. Reports of Biochemistry & Molecular Biology, 2020, Vol.8, No.4, http://rbmb.net/article-1-391-en.pdf3, Q3. Percentile – 46.
5. Mukanov, K.K., Adish, Z.B., Mukantayev, K.N., Tursunov, K.A., Kairova, Z.K., Kaukabayeva, G.K. Kulyyassov, A.T., Tarlykov, P.V. Recombinant expression and purification of adenocarcinoma gpr161 receptor. Asia-Pacific Journal of Molecular Biology and Biotechnology, 2019, 27(4), P.85–95. https://doi.org/10.35118/apjmbb.2019.027.4.10 Q4.
6. Khilyas, I.V., Tursunov, K.A., Shirshikova, T.V., Kamaletdinova, L.K., Matrosova, L.E., Desai, P. T., McClelland, M., Bogomolnaya, L.M. Genome Sequence of Pigmented Siderophore-Producing Strain Serratia marcescens SM6. Microbiology Resource Announcements, 2019, 8(18). https://doi.org/10.1128/mra.00247-19. Q4, Percentile – 29, Citations – 13.
7. Mukantayev K.N., Tursunov K.A., Kanayev D.B., Tokhtarova L., Ramankulov Ye. M., Mukanov K.K. Obtaining strain-producer of recombinant hexon of bovine adenovirus type 3. Eurasian Journal of Applied Biotechnology, 2019, Vol.1, 73-84. https://doi.org/10.11134/btp.1.2019.9.
8. Adish Zh., Mukantaev K.N., Tursunov K.A., Kaukabayeva G.K., Kanaev D.B., Ramankulov Ye.M., Mukanov K.K. Obtaining and determination of immunogenic properties of TRX-PD-1 recombinant protein. Eurasian Journal of Applied Biotechnology, 2019, Vol.1, 33-42. https://doi.org/10.11134/btp.1.2019.3.
9. Bulashev A., Jakubowski T., Mukantayev K., Tursunov K., Kiyan V., Zhumalin A. Using combined recombinant protein in the diagnosis of bovine brucellosis. Med. Weter. 2018, 74 (3), 193-198. www.doi.org/10.21521/mw.6079. Q3.
10. Bulashev, A., Jakubowski, T., Tursunov, K., Kiyan, V., Zhumalin, A. Immunogenicity and antigenicity of Brucella recombinant outer membrane proteins. Veterinarija Ir Zootechnika, 2018, 76(98), 17–24. https://vetzoo.lsmuni.lt/data/vols/2018/76/pdf/bulashev.pdf Q4.
11. Kanatbek Mukantayev, Kanat Tursunov, Guljan Raimbek, Alexander Shustov, Asem Begaliyeva, Bakhytkali Ingirbay, Kasym Mukanov, Erlan Ramanculov. Immunochromatographic assay for diagnosis of bovine leukaemia virus infection in cows using the recombinant protein gp51. ISSN 1392-2130. Veterinarija Ir Zootechnika (Vet Med Zoot). T. 2018, 76 (98). p. 34-40. https://vetzoo.lsmuni.lt/data/vols/2018/76/pdf/mukantayev.pdf. Q4.
12. Mukantayev, K., K. Tursunov, B. Ingirbay, Z. Adish, M. Azhibayeva, Z. Kairova, E. Ramankulov, K. Mukanov and A. Shustov. Immunochromatographic assay for the foot-and-mouth disease utilizing recombinant nonstructural proteins 2C, 3A, 3B and 3D. Bulgarian Journal Agricultural Science, 2018, 24 (3): 489–496. https://www.agrojournal.org/24/03-21.html. Q4. Percentile – 45.
13. Bulashev, A., Jakubowski, T., Tursunov, K., Kiyan, V., Zhumalin, A. Immunogenicity and antigenicity of Brucella recombinant outer membrane proteins. Veterinarija Ir Zootechnika, 2018, 76(98), 17–24. https://vetzoo.lsmuni.lt/data/vols/2018/76/pdf/bulashev.pdf Q4, Percentile – 23, Citations – 4.
14. Tursunov K., Begaliyeva A., Ingirbay B., Mukanov K., Ramanculov E., Shustov A., Mukantayev K. Cloning and expression of fragment of the rabies virus nucleoprotein gene in Escherichia coli and evaluation of antigenicity of the expression product. Iranian Journal of Veterinary Research. – 2017. –Vol.18, No. 1, Ser. No. 58, P.36-42. PMID: 28588631 PMCID: PMC5454577. Q3, Percentile – 45, Citations – 4.
15. Escherichia coli strain BL21(DE3)/pET32/NPRV, producer of recombinant rabies virus nucleoprotein. Author’s ID No. 101822. Mukantayev K.N., Shustov A.V., Tursunov K.A., Ingirbay B., Adish Zh., Ramankulov E.M., Mukanov K.K.
16. Escherichia coli strain BL21(DE3)/pET32/OmpBm-Ba, producer of recombinant chimeric protein of the outer membrane of Brucella. Author’s ID No. 104361. Bulashev A.K., Tursunov K.A., Zhumalin A.Kh., Mukantayev K.N.
17. Escherichia coli strain BL21(DE3) pET28a/Omp19/31, producer of a chimeric recombinant Brucella outer membrane protein. Patent for invention No. 35776. 07/29/2022. Bulashev A.K., Akibekov O.S., Ingirbay B.K., Mukantayev K.N., Syzdykova A.S., Suranshiev Zh.A., Tursunov K.A.
18. Genetic construct Pet28/CTLA-4, which ensures the production of the extracellular domain of the human CTLA-4 receptor in transformed E. coli. Patent for invention No. 36343. 08/18/2023. Mukantayev K.N., Mukanov K.K., Tursunov K.A., Adish Zh.B., Ramankulov E.M.
19. Genetic construct pET32/P32, designed to express the P32 gene of the bovine lumpy skin disease virus. Patent for invention No. 36373. 09/22/2023. Tursunov K.A., Mukantaev K.N., Kanayev D.B., Adish Zh.B., Ramankulov E.M.
2021
A genetic construct carrying the P32 antigen gene was obtained. The resulting DNA fragment was cloned into expression vectors, resulting in genetic constructs carrying the P32 gene. E. coli BL21 (DE3) strains were obtained as producers of the recombinant P32 antigen of the LSDV with a molecular weight of 47 kDa (rP32Trx/pET32) and 32 kDa (rP32/pET28). The main stages of isolation and purification of recombinant P32 protein have been worked out. The results of mass spectrometry (MS/MS) of recombinant proteins analyzed using the MASCOT program confirmed the presence of the P32 antigen of the LSDV.
2022
The immunological and diagnostic characteristics of the recombinant P32 antigen were studied. A scheme for immunization of laboratory animals with recombinant P32 protein has been developed. The main parameters for performing indirect ELISA have been optimized. Two lines of hybridoma cells producing monoclonal antibodies against the recombinant P32 antigen were obtained. Both hybrid cell lines (4E2A11 and 8F2E12) produce antibodies related to immunoglobulin class G, subclass 2b, light chain type – kappa. The binding constant of monoclonal antibodies produced by the hybridoma cell lines 4E2A11 and 8F2E12 was 3×107M-1 and 2×108M-1, respectively. When producing a preparative amount of monoclonal antibodies using the in vitro method, the antibody concentration averaged 43 μg/ml. Using the in vivo method, on Balb/c mice, an average of 4-5 mg/ml was obtained.
2023
The specificity of monoclonal antibodies obtained to the recombinant P32 antigen was determined. The main parameters for the use of recombinant P32 protein and monoclonal antibodies to the P32 antigen in immunochromatographic analysis have been worked out. It was found that the optimal concentration of protein G (pH 6.0) and monoclonal antibodies (pH 9.0) for immobilization on colloidal gold, with a particle size of 20 nm in diameter, was 20 μg/ml and 12 μg/ml, respectively. The optimal concentration of monoclonal antibodies, antigen and secondary antispecies antibodies applied to the membrane was 200 μg/ml, 500-800 μg/ml and 200-300 μg/ml, respectively. The conjugate and monoclonal antibodies specifically bound to the rP32 protein and did not cross-react with other proteins. The parameters for the formation of an immunochromatographic test were worked out and its diagnostic properties were determined. The sensitivity and specificity of the test were 79% and 90%, respectively.