Production of Monoclonal Antibody against Prokaryotically Expressed G1 Protein of Bovine Ephemeral Fever Virus
محورهای موضوعی : Camelر. پسندیده 1 , م.ر. صیفی آباد شاپوری 2 , م.ت. بیگی نصیری 3
1 - Department of Animal Science, Faculty of Animal and Food Science, Khuzestan Agricultural Science and Natural Resources University, Ahvaz, Iran
2 - Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
3 - Department of Animal Science, Faculty of Animal and Food Science, Khuzestan Agricultural Science and Natural Resources University, Ahvaz, Iran
کلید واژه: bovine ephemeral fever virus, hybridoma, monoclonal antibody, recombinant G1 protein,
چکیده مقاله :
Epitope-G1 of bovine ephemeral fever virus (BEFV) G glycoprotein has been genetically and antigenically conserved among various isolates of BEFV and only reacts with anti-BEFV neutralising antibodies. Therefore, it is a candidate antigen for development of the enzyme linked immunosorbent assay (ELISA) for serological identification bovine ephemeral fever (BEF)-infected animals. The aim of this study was to produce a monoclonal antibody (MAb) against recombinant G1 antigen expressed into Escherichia coli. For this purpose, somatic cell hybrids between SP2/0 myeloma cells and spleen cells derived from Balb/c mice immunized with maltose-binding protein (MBP)-G1 fusion protein were established. After three rounds of cloning, the stability of antibody secretion in the positive clones was confirmed by ELISA and the reactivity of the MAbs against recombinant G1 was verified by Western blot analysis. The specific MAbs produced against recombinant G1 antigen in this study could be used for establishing BEFV diagnostic experiments in the future.
اپیتوپ G1 از گلیکوپروتئین G ویروس تب بی دوام گاوی (BEFV) از لحاظ ژنتیکی و آنتی ژنیکی در بین جدایه های مختلف از این ویروس حفاظت شده است و تنها با آنتی بادی های خنثی کننده ضد این ویروس واکنش می دهد. بنابراین این آنتیژن، یک نامزد مناسب برای توسعه آزمون ایمونوسوربنت متصل به آنزیم (ELISA) برای شناسایی سرولوژیکی حیوانات آلوده است. هدف از این مطالعه، تولید آنتی بادی مونوکلونال علیه آنتیژن G1 نوترکیب بیان شده در اشرشیاکلی بود. به این منظور، هیبریدهای سلولی سوماتیک بین سلول های میلومای SP2/0 و سلول های طحال حاصل از موش های Balb/c ایمن شده با پروتئین هم جوش MBP-G1 ایجاد شد. پس از سه مرتبه کلونینگ، پایداری ترشح آنتی بادی درکلون های مثبت با روش الایزا و واکنش آنتی بادی های مونوکلونال علیه G1 نوترکیب توسط آزمایش وسترن بلات تأیید شد. آنتی بادی های اختصاصی تولید شده علیه آنتی ژن نوترکیب G1 ممکن است به منظور طراحی آزمایش های تشخیصی برای ویروس BEF در آینده مناسب باشند.
Ausubel F., Brent R., Kingston R., Moore D., Seidman J., Smith J. and Struhl K. (1992). Short Protocols in Molecular Biology. John WileyandSons,New York.
Aziz-Boaron O., Leibovitz K., Gelman B., Kedmi M. and Klement E. (2013). Safety, immunogenicity and duration of immunity elicited by an inactivated bovine ephemeral fever vaccine. PLoS One. 8, e82217.
Beygi Nassiri M.T., Pasandideh R. and Seyfi Abad Shapouri M.R. (2016). Cloning and Expression of the G1 Epitope of Bovine Ephemeral Fever Virus G Glycoprotein in Escherichia Coli. Genetics in the 3rdMillennium. 14, 4250-4255.
Cybinski D., Walker P., Byrne K. and Zakrzewski H. (1990). Mapping of antigenic sites on the bovine ephemeral fever virus glycoprotein using monoclonal antibodies. J. Gen. Virol. 71, 2065-2072.
Deb R., Chakraborty S., Veeregowda B., Verma A.K., Tiwari R. and Dhama K. (2013). Monoclonal antibody and its use in the diagnosis of livestock diseases. Adv. Biosci. Biotechnol. 4, 50-62.
Dhillon J., Cowley J.A., Wang Y. and Walker P.J. (2000). RNA polymerase (L) gene and genome terminal sequences of ephemeroviruses bovine ephemeral fever virus and Adelaide River virus indicate a close relationship to vesiculoviruses. Virus Res. 70, 87-95.
Eto N., Yamada K., Koga A., Shirahata S. and Murakami H. (1991). Establishment and characterization of monoclonal antibodies against bovine ephemeral fever virus. Agric. Biol. Chem. 55, 167-172.
Goding J.W. (1980). Antibody production by hybridomas. J. Immunol. Methods. 39, 285-308.
Harlow E. and Lane D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, New York
Kapust R.B. and Waugh D.S. (1999). Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8, 1668-1674.
Kato T., Aizawa M., Takayoshi K., Kokuba T., Yanase T., Shirafuji H., Tsuda T. and Yamakawa M. (2009). Phylogenetic relationships of the G gene sequence of bovine ephemeral fever virus isolated in Japan, Taiwan and Australia. Vet. Microbiol. 137, 217-223.
Kirkland P.D. (2002). Akabane and bovine ephemeral fever virus infections. Vet. Clin. North America: Food Anim. 18, 501-514.
Nandi S. and Negi B. (1999). Bovine ephemeral fever: A review. Comp. Immunol. Microbiol. 22, 81-91.
Pasandideh R., Seyfi Abad Shapouri M.R. and Beigi Nassiri M.T. (2018). Immunogenicity of a plasmid DNA vaccine encoding G1 epitope of bovine ephemeral fever virus G glycoprotein in mice. Onderstepoort J. Vet. Res. 85, 1-6.
Siddiqui M. (2010). Monoclonal antibodies as diagnostics; an appraisal. Indian J. Pharm. Sci. 72, 12-17.
St George T., Cybinski D., Murphy G. and Dimmock C.K. (1984). Serological and biochemical factors in bovine ephemeral fever. Australian J. Biol. Sci. 37, 341-350.
Stram Y., Kuznetzova L., Levin A., Yadin H. and Rubinstein-Giuni M. (2005). A real-time RT-quantative (q) PCR for the detection of bovine ephemeral fever virus. J. Virol. Methods. 130, 1-6.
Trinidad L., Blasdell K.R., Joubert D.A., Davis S.S., Melville L., Kirkland P.D., Coulibaly F., Holmes E.C. and Walker P.J. (2014). Evolution of bovine ephemeral fever virus in the Australian episystem. J. Virol. 88, 1525-1535.
Uren M., Walker P., Zakrzewski H., St George T. and Byrne K. (1994). Effective vaccination of cattle using the virion G protein of bovine ephemeral fever virus as an antigen. Vaccine. 12, 845-852.
Walker P.J. and Klement E. (2015). Epidemiology and control of bovine ephemeral fever. Vet. Res. 46, 1-19.
Wallace D.B. and Viljoen G.J. (2005). Immune responses to recombinants of the South African vaccine strain of lumpy skin disease virus generated by using thymidine kinase gene insertion. Vaccine. 23, 3061-3067.
Yin Z. and Liu J.H. (1997). Animal Virology. Science Press, Beijing, China.
Zakrzewski H., Cybinski D. and Walker P. (1992). A blocking ELISA for the detection of specific antibodies to bovine ephemeral fever virus. J. Immunol. Methods. 151, 289-297.
Zheng F.Y. and Qiu C.Q. (2012). Phylogenetic relationships of the glycoprotein gene of bovine ephemeral fever virus isolated from mainland China, Taiwan, Japan, Turkey, Israel and Australia. J. Virol. 9, 1-8.
Zheng F.Y., Lin G.Z., Zhou J.Z., Wang G., Cao X.A., Gong X.W. and Qiu C.Q. (2011). A reverse-transcription, loop-mediated isothermal amplification assay for detection of bovine ephemeral fever virus in the blood of infected cattle. J. Virol. Methods. 171, 306-309.
Zheng F.Y., Lin G.Z., Qiu C.Q., Zhou J.Z., Cao X.A. and Gong X.W. (2009). Development and application of G 1-ELISA for detection of antibodies against bovine ephemeral fever virus. Res. Vet. Sci. 87, 211-212.
Zheng F.Y., Lin G.Z., Qiu C.Q., Zhou J.Z., Cao X.A. and Gong X.W. (2010). Serological detection of bovine ephemeral fever virus using an indirect ELISA based on antigenic site G1 expressed in Pichia pastoris. Vet. J. 185, 211-215.
