Cloning genes of p49, p72, pe199l, pe248r and cd2vinto Pichia Pastoris gs115
Author affiliations
DOI:
https://doi.org/10.15625/vjbt-21562Keywords:
CD2v, p49, p72, pE199L, pE248R, Pichia pastoris GS115, pPIC9K plasmid, African swine fever virusAbstract
African swine fever (ASF), caused by the African swine fever virus (ASFV), poses a significant threat to pig populations worldwide. ASFV is a double-stranded DNA virus. In recent years, 54 structural proteins and more than 100 proteins have been involved in viral infection found in macrophages of diseased pigs. Among these, p49 and p72 are essential capsid proteins crucial for forming the viral capsid. Additionally, pE199L and pE248R, located in the inner viral membrane, are critical for membrane fusion, a necessary step for viral entry into host cells. Another important player is CD2v, a type I transmembrane protein involved in the infection process. In this research, to develop subunit vaccines against ASFV, we focused on cloning the genes encoding these five proteins—p49, p72, pE199L, pE248R, and CD2v—into the pPIC9K plasmid for expression in the Pichia pastoris GS115 yeast strain. The viral genomic DNA was extracted from blood samples of infected pigs, and the genes encoding the five proteins were successfully amplified using Phusion PCR. The PCR products of each gene were then digested with EcoRI and NotI restriction enzymes and ligated into the pPIC9K plasmid. After that, we transformed the recombinant plasmids into Escherichia coli DH5α for amplification and purification. The plasmids were subsequently linearized with SalI and introduced into P. pastoris GS115 through electroporation. The selection of appropriate media and PCR analysis of the genomic DNA confirmed the successful generation of five recombinant P. pastoris GS115 strains. This work paves the way for the development of a recombinant protein vaccine against ASF by using the Pichia pastoris GS115 in the future.
Downloads
References
Arias, M., De la Torre, A., Dixon, L., Gallardo, C., Jori, F., Laddomada, A., Martins, C., Parkhouse, R. M., Revilla, Y., Rodriguez, F. and, & Sanchez-Vizcaino. (2017). Approaches and perspectives for development of African swine fever virus vaccines. Vaccines, 5(4), 35. https://doi.org/10.3390/vaccines5040035
Cwynar, P., Stojkov, J., & Wlazlak, K. (2019). African swine fever status in Europe. Viruses, 11(4), 310. https://doi.org/10.3390/v11040310
de Villiers, E. P., Gallardo, C., Arias, M., da Silva, M., Upton, C., Martin, R., & Bishop, R. P. (2010). Phylogenomic analysis of 11 complete African swine fever virus genome sequences. Virology, 400(1), 128–136. https://doi.org/10.1016/j.virol.2010.01.019
Dixon, L. K., Chapman, D. A. G., Netherton, C. L., & Upton, C. (2013). African swine fever virus replication and Genomics. Virus Research, 173(1), 3–14. https://doi.org/10.1016/j.virusres.2012.10.020
Ferrer-Miralles, N., & Villaverde, A. (2013). Bacterial cell factories for recombinant protein production; expanding the catalogue. Microbial Cell Factories, 12(1), 113. https://doi.org/10.1186/1475-2859-12-113
Gómez-Puertas, P., Rodríguez, F., Oviedo, J. M., Ramiro-Ibáñez, F., Ruiz-Gonzalvo, F., Alonso, C., & Escribano, J. M. (1996). Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization. Journal of Virology, 70(8), 5689–5694. https://doi.org/10.1128/jvi.70.8.5689-5694.1996
Kalenzi Atuhaire, D., Ochwo, S., Afayoa, M., Norbert Mwiine, F., Kokas, I., Arinaitwe, E., Ademun-Okurut, R. A., Boniface Okuni, J., Nanteza, A., Ayebazibwe, C., Okedi, L., Olaho-Mukani, W., & Ojok, L. (2013). Epidemiological overview of African Swine Fever in Uganda (2001–2012). Journal of Veterinary Medicine, 2013, 1–9. https://doi.org/10.1155/2013/949638
King, K., Chapman, D., Argilaguet, J. M., Fishbourne, E., Hutet, E., Cariolet, R., Hutchings, G., Oura, C. A. L., Netherton, C. L., Moffat, K., Taylor, G., Le Potier, M.-F., Dixon, L. K., & Takamatsu, H.-H. (2011). Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunisation. Vaccine, 29(28), 4593–4600. https://doi.org/10.1016/j.vaccine.2011.04.052
Li, Z., Chen, W., Qiu, Z., Li, Y., Fan, J., Wu, K., Li, X., Zhao, M., Ding, H., Fan, S., & Chen, J. (2022). African swine fever virus: A Review. Life, 12(8), 1255. https://doi.org/10.3390/life12081255
Lopera-Madrid, J., Medina-Magües, L. G., Gladue, D. P., Borca, M. V., & Osorio, J. E. (2021). Optimization in the expression of ASFV proteins for the development of subunit vaccines using poxviruses as delivery vectors. Scientific Reports, 11(1). https://doi.org/10.1038/s41598-021-02949-x
Neilan, J. G., Zsak, L., Lu, Z., Burrage, T. G., Kutish, G. F., & Rock, D. L. (2004). Neutralizing antibodies to African swine fever virus proteins P30, P54, and P72 are not sufficient for antibody-mediated protection. Virology, 319(2), 337–342. https://doi.org/10.1016/j.virol.2003.11.011
Nguyễn, B. T., Phạm , L. T., & Nguyễn , P. T. (2023). Nhân Dòng, Biểu Hiện và Tinh Sạch Protein Interleukin-2 Của Người (Il-2) Hướng Đến Ứng Dụng Chữa Lành Vết Thương. Hội Nghị Công Nghệ Sinh Học Toàn Quốc 2023, 426–431.
Salas, M. L., & Andrés, G. (2013). African swine fever virus morphogenesis. Virus Research, 173(1), 29–41. https://doi.org/10.1016/j.virusres.2012.09.016
Tran, H. T., Truong, A. D., Ly, D. V., Vu, T. H., Hoang, V. T., Nguyen, T. C., Chu, T. N., Nguyen, T. H., Pham, N. T., Nguyen, T., Yersin, A. G., & Dang, H. V. (2020). Genetic characterisation of African swine fever virus in outbreaks in Ha Nam Province, Red River Delta region of Vietnam, and activity of antimicrobial products against virus infection in contaminated feed. Journal of Veterinary Research, 64(2), 207–213. https://doi.org/10.2478/jvetres-2020-0041
Tran, M. N., Nguyen, H. M., Le, L. T., Doan, H. T., Nguyen, M. M., Dinh, P. X., & Nguyen, B. T. (2023). Sequencing P72 gene of field strain of African swine fever virus (ASFV) in Vietnam and generation of enhanced immunogenic fusion protein G-P72 potentially expressed as a recombinant antigen in ASFV subunit vaccine. The Journal of Agriculture and Development, 22(06), 32–41. https://doi.org/10.52997/jad.4.06.2023
Wu, S., & Letchworth, G. J. (2004). High efficiency transformation by electroporation of pichia pastoris pretreated with lithium acetate and Dithiothreitol. BioTechniques, 36(1), 152–154. https://doi.org/10.2144/04361dd02
Yao, J.-Y., Zhang, C.-S., Yuan, X.-M., Huang, L., Hu, D.-Y., Yu, Z., Yin, W.-L., Lin, L.-Y., Pan, X.-Y., Yang, G., Wang, C.-F., Shen, J.-Y., & Zhang, H.-Q. (2022). Oral vaccination with recombinant pichia pastoris expressing iridovirus major capsid protein elicits protective immunity in largemouth bass (Micropterus salmoides). Frontiers in Immunology, 13. https://doi.org/10.3389/fimmu.2022.852300
Yoon, H., Hong, S., Lee, I., Yoo, D., Jung, C., Lee, E., & Wee, S. (2020). Clinical symptoms of African swine fever in domestic pig farms in the Republic of Korea, 2019. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.13552
Zhang, G., Liu, W., Gao, Z., Chang, Y., Yang, S., Peng, Q., Ge, S., Kang, B., Shao, J., & Chang, H. (2022). Antigenic and immunogenic properties of recombinant proteins consisting of two immunodominant African swine fever virus proteins fused with bacterial lipoprotein opri. Virology Journal, 19(1). https://doi.org/10.1186/s12985-022-01747-9
Downloads
Published
How to Cite
Issue
Section
