The main idea of the project is to develop an enzyme-linked immunosorbent assay based on a recombinant chimeric Toxoplasma gondii protein consisting of immunodominant sequences of the three most promising antigens for the serological diagnosis of toxoplasmosis in farm animals. The project is aimed at solving the problems associated with the spread of toxoplasmosis among farm animals, which are the main source of human infection. The enzyme-linked immunosorbent assay based on a recombinant chimeric protein will increase the efficiency of diagnosis and improve the epizootological situation of toxoplasmosis.
Toxoplasmosis is a widespread zoonotic infection caused by the intracellular parasite Toxoplasma gondii, which affects almost all warm-blooded animals, as well as humans. Toxoplasmosis affects about a third of the world’s population. This is of great economic and social importance, affecting public health and livestock production. Human infection with toxoplasmosis occurs when eating raw or undercooked meat of infected animals, as well as food or water contaminated with oocysts. The main spreaders of the parasite are cats. They shed millions of parasitic oocysts in their faeces into the environment, which after sporulation can retain their infectivity for a long time in water and soil and are responsible for infecting intermediate hosts, including humans and other animals. In general, the disease is asymptomatic or may present with flu-like symptoms and other nonspecific clinical signs. However, in immunocompromised patients, the disease can be severe and even fatal. This disease is especially dangerous for pregnant women, as it is transmitted vertically from mother to fetus through the placenta. The infection can lead to serious neurological or ocular disorders with heart and brain abnormalities such as hydrocephalus, mental retardation, seizures, fetal death, and abortion. In farm animals, in particular sheep and goats, infection can lead to abortion, mummification or maceration of the fetus, intrauterine embryonic death, stillbirth or postnatal death of newborns, which threatens sheep and goat farming worldwide. Surviving offsprings are often weak and susceptible to other diseases. The productivity of farm animals largely depends on their reproductive efficiency. High fetal mortality caused by a wide range of protozoa, bacteria and viruses is the main cause of economic losses in the agricultural industry and ruminant farms.
In 2012, according to a report published by the Food Standards Agency’s Microbiological Safety Advisory Committee, human toxoplasmosis was the second leading cause of foodborne disease death in the United States, accounting for 24% of all deaths compared to 28% for Salmonella spp. At the same time, the consumption of sheep meat was the cause of 30% of human diseases. It should be noted that there are no data on the epizootology of toxoplasmosis in cattle and small ruminants in Kazakhstan, while the meat of these animals is widely consumed in the country and may represent an important source of transmission of toxoplasmosis to humans.
As a result of the development of genetic engineering, today a completely new approach has appeared in the construction of recombinant products, the so-called chimeric antigens, which can replace native antigens from a lysed whole parasite. Chimeric recombinant antigens may contain different immunoreactive epitopes of several T. gondii antigens. The advantage of chimeric recombinant antigens over individual recombinant antigens is the higher sensitivity of immunological tests and lower cost of the final product. This is due to the fact that the production of one chimeric recombinant antigen carrying the immunodominant regions of three proteins will require significantly less costs compared to obtaining three recombinant proteins separately. The scientific novelty of the project lies in the development of an ELISA based on recombinant chimeric antigens for the detection of anti-T. gondii IgG in the serum of farm animals (cows, sheep and goats).
The aim of the project is to develop an enzyme-linked immunosorbent assay based on a recombinant chimeric protein consisting of a combination of the three most promising antigens for the diagnosis of toxoplasmosis in cows, sheep and goats.
In accordance with the goal and objectives of the project, an ELISA based on a recombinant chimeric antigen will be developed for the diagnosis of toxoplasmosis in farm animals. At the first stage of research, the analysis of known immunodominant antigens of T. gondii will be carried out and the three most promising proteins will be selected. Further, strains of microorganisms producing the recombinant chimeric antigen will be obtained and the parameters of its isolation and purification will be worked out. At the next stage, the preparative production of the native Toxoplasma lysate antigen will be carried out. The biochemical properties of the obtained recombinant chimeric antigen will be studied using nanoLC-MS/MS spectrometry methods. The immunological and diagnostic properties of proteins will be studied. At the final stage, the main parameters of the ELISA will be worked out and optimized. The efficiency of the prepared ELISA will be studied.
Tursunov Kanat Akhmetovich, PhD, H-index 3 (Author ID Scopus – 57193579180, Researcher ID Web of Science N-6319-2017)
Mukantayev Kanatbek Naizabekovich, higher education, Doctor of Biological Sciences, Associate Professor. h-индекс 4, Researcher ID: AAM-8674-2020; ORCID: 0000-0002-6048-0232; Author ID Scopus: 57211138932.
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. 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 – 10.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
2023
A genetic construct based on the pGEM-T vector was obtained, carrying the amino acid sequence of the SAG1, SAG2 and GRA7 proteins of T. gondii. For this purpose, ten T. gondii proteins were selected from the NCBI database and analyzed. Next, a genetic construct was obtained based on the expression vector pET28, carrying the gene for the chimeric protein of the T. gondii parasite. The resulting recombinant plasmid was transformed into E. coli BL21(DE3) cells by electroporation. Positive colonies were grown in large volumes on a nutrient medium and then the expression of the recombinant protein was determined. It was found that the most optimal IPTG concentration for protein expression is 0.2 mM, the incubation temperature is 37°C, and the optimal incubation time is 16-18 hours. The resulting strain produces a recombinant chimeric protein with a molecular weight of about 38 kDa. Methods for isolating and purifying the recombinant chimeric protein were developed.