Expression of recombinant classical swine fever virus E2 glycoprotein instable High Five cells
![](/public/icon/gor.png)
![](/public/icon/pdf.png)
![](/public/icon/pdf.png)
Keywords:
Classical swine fever virus (CSFV), Dot blot, Insect cell expression, Western blotAbstract
Classical swine fever is one of the most important viral diseases of domestic pigs and wild boar. It is a notifiable disease to the World Organization for Animal Health. In the present study, codon optimized CSFV E2 gene was cloned into a laboratory-modified insect cell expression vector for producing the recombinant protein in secretory form. Endotoxin-free recombinant plasmid prepared from the positive clone was transfected into the High Five insect cell line and then selected against zeocin antibiotic for the development of a stable cell line. The stable High Five cell line successfully secreted E2 recombinant protein with a molecular weight of ~65 kDa as revealed by SDS-PAGE and Western blot. The recombinant E2 protein showed efficient reactivity with known CSFV anti-serum and monoclonal antibodies (MAbs against E2) in dot blot assay indicating its potential as a diagnostic antigen.
Downloads
References
Amer H M. 2011. Baculovirus expression vector system: An efficient tool for the production of heterologous recombinant proteins. African Journal of Biotechnology 10(32): 5927–33.
Backliwal G, Hildinger M, Chenuet S, Wulhfard S, De Jesus M and Wurm F M. 2008. Rational vector design and multi- pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Research 36(15): e96– e96.
Borca M V, Holinka L G, Ramirez-Medina E, Risatti G R, Vuono E A, Berggren K A and Gladue D P. 2019. Identification of structural glycoprotein E2 domain critical to mediate replication of Classical Swine Fever Virus in SK6 cells. Virology 526: 38–44.
Daramola O, Stevenson J, Dean G, Hatton D, Pettman G, Holmes W and Field R. 2014. A high‐yielding CHO transient system: Coexpression of genes encoding EBNA‐1 and GS enhances transient protein expression. Biotechnology Progress 30(1): 132–41.
Elbers K, Tautz N, Becher P, Stoll D, Rümenapf T and Thiel H J. 1996. Processing in the pestivirus E2-NS2 region: Identification of proteins p7 and E2p7. Journal of Virology 70(6): 4131–35.
Harrison R L and Jarvis D L. 2007. Transforming lepidopteran insect cells for improved protein processing, pp. 341–56. (Ed) Murhammer D W. Baculovirus and Insect Cell Expression Protocols. Methods in Molecular Biology. Vol. 388. Humana Press.
Hua R H, Huo H, Li Y N, Xue Y, Wang X L, Guo L P, Zhou B, Song Y and Bu Z G. 2014. Generation and efficacy evaluation of recombinant classical swine fever virus E2 glycoprotein expressed in stable transgenic mammalian cell line. PLoS One 9(9): e106891.
Ito S, Jurado C, Bosch J, Ito M, Sánchez-Vizcaíno J M, Isoda N and Sakoda Y. 2019. Role of wild boar in the spread of classical swine fever in Japan. Pathogens 24(4): 206.
Krammer F, Schinko T, Palmberger D, Tauer C, Messner P and Grabherr R. 2010. Trichoplusia ni cells (High Five TM) are highly efficient for the production of influenza A virus-like particles: A comparison of two insect cell lines as production platforms for influenza vaccines. Molecular Biotechnology 45: 226–34.
Li L, Weinberg C R, Darden T A and Pedersen L G. 2001. Gene selection for sample classification based on gene expression data: study of sensitivity to choice of parameters of the GA/ KNN method. Bioinformatics 17(12): 1131–42.
Loomis K H, Yaeger K W, Batenjany M M, Mehler M M, Grabski A C, Wong S C and Novy R E. 2005. Insect Direct TM System: Rapid, high-level protein expression and purification from insect cells. Journal of Structural and Functional Genomics 6: 189–94.
Madera R F, Wang L, Gong W, Burakova Y, Buist S, Nietfeld J, Henningson J, Cino-Ozuna A G, Tu C and Shi J. 2018. Toward the development of a one-dose classical swine fever subunit vaccine: antigen titration, immunity onset, and duration of immunity. Journal of Veterinary Science 19(3): 393–405.
Maurer R, Stettler P, Ruggli N, Hofmann M A and Tratschin J D. 2005. Oronasal vaccination with classical swine fever virus (CSFV) replicon particles with either partial or complete deletion of the E2 gene induces partial protection against lethal challenge with highly virulent CSFV. Vaccine 23(25): 3318–28.
Moennig V. 2000. Introduction to classical swine fever: Virus, disease and control policy. Veterinary Microbiology 73(2-3): 93–102.
Ogay I D, Lihoradova O A, Azimova S S, Abdukarimov A A, Slack J M and Lynn D E. 2006. Transfection of insect cell lines using polyethylenimine. Cytotechnology 51: 89–98.
Patil S S, Suresh K P, Hemadri D, Hiremath J, Sridevi R, Krishnamoorthy P, Bhatia S and Roy P. 2021.Spatial seroprevalence of classical swine fever in India. Tropical Animal Health and Production 53(3): 389.
Postel A, Nishi T, Kameyama K I, Meyer D, Suckstorff O, Fukai K and Becher P. 2019. Re-emergence of classical swine fever, Japan. Emerging Infectious Diseases 25(6): 1228.
Righi C, Petrini S, Pierini I, Giammarioli M and De Mia G M. 2021. Global distribution and genetic heterogeneity of border disease virus. Viruses 13(6): 950.
Shen X, Hacker D L, Baldi L and Wurm F M. 2014. Virus- free transient protein production in Sf9 cells. Journal of Biotechnology 171: 61–70.
Shen X, Pitol A K, Bachmann V, Hacker D L, Baldi L and Wurm F M. 2015. A simple plasmid-based transient gene expression method using High Five cells. Journal of Biotechnology 216: 67–75.
Van Gennip H G, Bouma A, Van Rijn P A, Widjojoatmodjo M N and Moormann R J. 2002. Experimental non-transmissible marker vaccines for classical swine fever (CSF) by trans- complementation of Erns or E2 of CSFV. Vaccine 20(11-12): 1544–56.
Van Rijn P A, Bossers A, Wensvoort G and Moormann R J. 1996. Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge. Journal of General Virology 77(11): 2737–45.
Vaughn J L, Goodwin R H, Tompkins G J and McCawley P J. 1977.The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In vitro 13(4): 213–7.
Wickham T J, Davis T, Granados R R, Shuler M L and Wood H A. 1992.Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotechnology Progress 8(5): 391–6.
Wilde M, Klausberger M, Palmberger D, Ernst W and Grabherr R. 2014. Tna o38, high five and Sf 9 evaluation of host–virus interactions in three different insect cell lines: Baculovirus production and recombinant protein expression. Biotechnology Letters 36: 743–9.
Downloads
Submitted
Published
Issue
Section
License
Copyright (c) 2024 The Indian Journal of Animal Sciences
![Creative Commons License](http://i.creativecommons.org/l/by-nc-sa/4.0/88x31.png)
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
The copyright of the articles published in The Indian Journal of Animal Sciences is vested with the Indian Council of Agricultural Research, which reserves the right to enter into any agreement with any organization in India or abroad, for reprography, photocopying, storage and dissemination of information. The Council has no objection to using the material, provided the information is not being utilized for commercial purposes and wherever the information is being used, proper credit is given to ICAR.