Bovine Ephemeral Fever Virüsün Rekombinant Glikoprotein ve Nükleoproteinlerinin Üretilmesi*

Öz

Bu çalışmada, bovine ephemeral fever virüsün (Üç gün hastalığı virüsü; BEFV) glikoprotein (G) ve nükleoprotein-lerinin (N) rekombinant DNA tekniği ile üretilmesi amaçlanmıştır. Bu amaçla, Türkiye izolatlarından çoğaltılan BEFV G ve N genlerini taşıyan pcDNA4/HisMax-TOPO ökaryotik ekspresyon vektörleri oluşturulmuş ve elde edilen rekombinant vektörlerle transfekte edilen hücrelerde bu proteinlerin in vitro üretilmesi gerçekleştirilmiştir. Geçici ve stabil transfekte hücrelerde RT-PCR ile mRNA düzeyinde genlerin transkripsiyonu doğrulanmıştır. Ayrıca, transfekte hücrelerden pürifi-ye edilen rekombinant proteinler Western blotlama deneyi ile gösterilmiştir. Bu çalışma sonucunda, BEFV teşhis ve korunmasında iki önemli proteinin üretimi ve pürifikasyonu gerçekleştirilmiştir. Elde edilen bu proteinlerin teşhis kitleri-nin hazırlanması ve sub-ünit aşı olarak kullanım potansiyeli bulunmaktadır.

Anahtar Kelimeler

• BEFV glikoprotein,BEFV nükleoprotein,bovine ephemeral fever,rekombinant DNA teknolojisi

Production of Recombinant Glycoprotein and Nucleoprotein of Bovine Ephemeral Fever Virus

Öz

This study is aimed to create a recombinant glycoprotein (G) and nucleoprotein (N) of bovine ephemeral fever virus (BEFV) by using DNA technology. For this purpose, G and N gene of BEFV Turkish isolate were cloned in to pcDNA4/HisMax-TOPO eukaryotic expression vector. Obtained recombinant vectors were transfected in Vero E6 cell line to produce recombinant protein. Presence of recombinant N and G gene of BEFV in Vero E6 was confirmed by using RT-PCR following transient transfection. Additionally purified recombinant N and G proteins of BEFV from Vero E6 were shown through Western blotting after stable transfection. With the help this study, we have achieved purifica-tion and in production of two recombinant proteins which play important role protection and diagnosis of BEFV. The obtained recombinant proteins will be used to create a sub-unit vaccine and to produce diagnosis kit for BEFV in future.

Anahtar Kelimeler

• BEFV glycoprotein,BEFV nucleoprotein,bovine ephemeral fever,recombinant DNA technology

Kaynakça

1. 1. Aziz-Boaron O, Klausner Z, Hasoksuz M, Shenkar J, Gafni O, Gelman B, David D, Klement E. Circulation of bovine ephemeral fever in the Middle East strong evidence for transmission by winds and animal transport. Vet Microbiol 2012; 158(4): 300-7. 2. Aziz-Boaron O, Gleser D, Yadin H, Gelman B, Kedmi M, Galon N, Klement E. The pro-tective effectiveness of an inactivated bovi-ne ephemeral fever virus vaccine. Vet Mic-robiol 2014; 173(2): 1-8. 3. Baranowska M, Hauge AG, Hoornaert C, Bogen B. Grødeland G. Targeting of nucle-oprotein to chemokine receptors by DNA vaccination results in increased CD8(+)-mediated cross protection against influen-za.Vaccine 2015; 33(49): 6988-96. 4. Barefoot BE, Sample CJ, Ramsburg EA. Recombinant vesicular stomatitis virus expressing influenza nucleoprotein induces CD8 T-cell responses that enhance antibody-mediated protection after lethal challenge with influenza virus. Clin Vaccine Immunol. 2009; 16(4): 488-98. 5. Barigye R, Melville LF, Davis S, Walsh S, Hunt N, Hunt R, Elliot N. Kinetics of pro- inflammatory cytokines, interleukin-10, and virus neutralising antibodies during acute ephemeral fever virus infections in Brahman cattle. Vet Immunol Immunopathol 2015; 168(4): 159-63. 6. Dietzschold B, Rupprecht CE, Tollis M, La-fon M, Mattei J, Wiktor TJ, Koprowski H. Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related viruses: implications for epidemio-logy and control of rabies. Rev Infect Dis, 1988; 4(2): 785-98. 7. Fujii H, Takita-Sonoda Y, Mifune K, Hirai K, Nishizono A, Mannen K. Protective efficacy in mice of post-exposure vaccination with vaccinia virus recombinant expressing either rabies virus glycoprotein or nucleoprotein. J Gen Virol 1994; 75(6): 1339-44. 8. Hertig C, Pye AD, Hyatt AD, Davis SS, McWilliam SM, Heine HG, Walker PJ, Boyle DB. Vaccinia virus-expressed bovine ephe-meral fever virus G but not G(NS) glycopro-tein induces neutralizing antibodies and pro-tects against experimental infection. J Gen Virol 1995; 77(4): 631-40. 9. Hsieh YC, Wang SY, Lee YF, Chen SH, Mak PO, Chu CY. DNA sequence analysis of glycoprotein G gene of bovine ephemeral fever virus and development of a double oil emulsion vaccine against bovine ephemeral fever. J Vet Med Sci 2006; 68(6): 543-8. 10. Johal J, Gresty K, Kongsuwan K, Walker PJ. Antigenic characterization of bovine ephe-meral fever rhabdovirus G and GNS glycop-roteins expressed from recombinant baculo-viruses. Arch Virol 2008; 153(9): 1657-65. 11. MacLachlan NJ, Dubovi EJ. Rhabdoviridae. Maclachlan NJ. Dubovi EJ. eds. In: Fenner’s Veterinary Virology. Fourth Edition. New York: Academic Press, 2011; pp. 338-49. 12. Nandi S, Negi BS. Bovine ephemeral fever:a review. Comp Immunol Microb 1999; 22(2): 81-91. 13. Nezam FS, Hosseini SM, Kheiri MT, Abdoli A. Memarnejadian A, Shenagari M, Gholami S, Sohani H, Rahmatollahi H, Jamali A. Suppressive effects of chronic stress on influenza virus protection after vaccination with plasmid DNA-encoded nucleoprotein. Neuroimmunomodul 2015; 22(5): 322-7. 14. Puddington L, Bevan MJ, Rose JK, Lefran-çois L. N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells. J Virol 1986; 60(2): 708-17. 15. St George TD, Murphy GM, Burren B, Uren MF. Studies on the pathogenesis of bovine ephemeral fever. IV: A comparison with the inflammatory events in milk fever of cattle. Vet Microbiol 1995; 46(1-3): 131-42. 16. Tonbak S, Berber E, Yoruk MD, Azkur AK, Pestil Z, Bulut H. A large-scale outbreak of bovine ephemeral fever in Turkey. J Vet Med Sci 2013; 75(11): 1511-4. 17. Sambrook J, Russell DW, eds. Molecular Cloning a Laboratory Manual. Third Edition. New York: Cold Spring Harbor Laboratory Press, 2001; p. 1-127. 18. Uren MF, Walker PJ, Zakrzewski H, St Ge-orge TD, Byrne KA. Effective vaccination of cattle using the virion G protein of bovine ephemeral fever virus as an antigen. Vacci-ne 1994; 12(9): 845-50. 19. Vanselow BA, Abetz I, Trenfield K. A bovine ephemeral fever vaccine incorporating adju-vant Quil A: a comparative study using adju-vants Quil A, aluminium hydroxide gel and dextran sulphate. Vet Rec 1985; 117(2): 37-43. 20. Venter GJ, Hamblin C, Paweska JT. Deter-mination of the oral susceptibility of South African livestock-associated biting midges, Culicoides species, to bovine ephemeral fever virus. Med Vet Entomol 2003; 17(2): 133-7. 21. Walker PJ, Byrne KA, Cybinski DH, Doolan DL, Wang YH. Proteins of bovine ephemeral fever virus. J Gen Virol 1991; 72(1): 67-74. 22. Walker PJ, Wang Y, Cowley JA, McWilliam SM, Prehaud CJ. Structural and antigenic analysis of the nucleoprotein of bovine ep-hemeral fever virus. J Gen Virol 1994; 75(8): 1889-99. 23. Wallace DB, Viljoen GJ. Immune responses to recombinants of the South African vacci-ne strain of lumpy skin disease virus gene-rated by using thymidine kinase gene inser-tion. Vaccine 2005; 23(23): 3061-7. 24. Yeruham I, Van Ham M, Bar D, Yadin H, Tiomkin D. Economic aspects of the 1999 outbreak of bovine ephemeral fever in dairy cattle herds in the Jordan Valley in Israel. Vet Rec 2003; 153(6): 180-2. 25. Zheng FY, Lin GZ, Qiu CQ, Zhou JZ, Cao XA, Gong XW. Development and application of G1-ELISA for detection of antibodies aga- inst bovine ephemeral fever virus. Res Vet Sci 2009; 87(2): 211-2. 26. Zheng FY, Lin GZ, Qiu CQ, Zhou JZ, Cao XA, Gong XW Serological detection of bovi-ne ephemeral fever virus using an indirect ELISA based on antigenic site G1 expres-sed in Pichia pastoris. Vet J 2010; 185(2): 211-5. 27. Zheng F, Qiu C. Phylogenetic relationships of the glycoprotein gene of bovine epheme-ral fever virus isolated from mainland China, Taiwan, Japan, Turkey, Israel and Australia. Virol J 2012; 9 (4): 268-75.