OBTAINING THE RECOMBINANT IMMUNOGENIC DOMAIN OF THE RABIES VIRUS NUCLEOPROTEIN
Main Article Content
Authors
K. Tursunov
National Center for Biotechnology, 13/5, Korgalzhyn rd., Astana, 010000, Kazakhstan
A. Begaliyeva
National Center for Biotechnology, 13/5, Korgalzhyn rd., Astana, 010000, Kazakhstan
B. Inirbay
National Center for Biotechnology, 13/5, Korgalzhyn rd., Astana, 010000, Kazakhstan
K.К. Mukanov
National Center for Biotechnology, 13/5, Korgalzhyn rd., Astana, 010000, Kazakhstan
Е.М. Ramanculov
National Center for Biotechnology, 13/5, Korgalzhyn rd., Astana, 010000, Kazakhstan
А.В. Shustov
National Center for Biotechnology, 13/5, Korgalzhyn rd., Astana, 010000, Kazakhstan
K.N. Mukantayev
National Center for Biotechnology, 13/5, Korgalzhyn rd., Astana, 010000, Kazakhstan
Abstract
Rabies is an important concern in the Republic of Kazakhstan due to its widespread incidence in the country. The main sources of the disease in the Republic are natural, such as viral transmission through populations of wild carnivores and rodents, and anthropurgic, such as viral transmission through populations of domesticated animals. The ability of rabies virus to spread rapidly underscores the importance of developing diagnostic measures during both prevention and quarantine efforts as these could be used to quell emerging out breaks. With the increase in the rate of diagnostic method development, problems have arisen with the inconsistency of antigens and antibodies used to develop diagnostic test systems. The rabies virus nucleoprotein (N protein) is an important target for the immunochemical diagnosis of rabies. Thus, we created a genetic construct based on plasmid pET32 that expresses the С-terminal fragment of the rabies virus nucleoprotein. Our genetic construct was transformed into E. coli competent cells and the parameters of isolation and purification of the recombinant rabies virus nucleoprotein were optimized. We found that the fragment with the most immunogenic site on the nucleoprotein was located at position 360–389 amino acids. The molecular weight of the recombinant antigen was 42 kDa. The serum from mice immunized with fixed rabies virus reacted specifically with this recombinant C-terminal fragment of the nucleoprotein virus. The serum from mice immunized with recombinant antigen and fixed virus showed cross-reactivity at high dilution titers.
Keywords
rabies virus, nucleoprotein, recombinant antigen, genetic construct, immunogenic domain, producing strains
Article Details
References
Masatani T., Ito N., Shimizu K., Ito Y., Nakagawa K., Sawaki Y., Koyama H., Sugiyama M. Rabies virus nucleoprotein functions to evade activation of the RIG-I-mediated antiviral response. Journal of Virology, 2010, vol. 84, pp. 4002-4012. PMID: 20130065.
Schoehn G., Iseni F., Mavrakis M., Blondel D., Ruigrok R.W.H. Structure of recombinant rabies virus nucleoprotein-RNA complex and identification of the phosphoprotein binding site. Journal of Virology, 2001, vol. 75, pp. 490-498. doi: 10.1128/JVI.75.1.490-498.2001.
Kawai A., Toriumi H., Tochikura T.S., Takahashi T., Honda Y., Morimoto K. Nucleocapsid formation and/or subsequent conformational change of rabies virus nucleoprotein (N) is a prerequisite step for acquiring the phosphatase-sensitive epitope of monoclonal antibody 5-2-26. Virology, 1999, vol. 263, pp. 395-407. PMID: 10544112.
Manjunatha Reddy G.B., Singh R., Singh R.P., Singh K.P., Gupta P.K., Desai A., Shankar S.K., Ramakrishnan M.A., Verm R. Molecular characterization of Indian rabies virus isolates by partial sequencing of nucleoprotein (N) and phosphoprotein (P) genes. Virus Genes, 2011, vol. 43, pp. 13-17. doi: 10.1007/s11262-011-0601-0.
Flamand A., Wiktor T.J., Koprowski H. Use of hybridoma monoclonal antibodies in the detection of antigenic differences between rabies and rabies-related virus proteins. I. The nucleocapsid protein. Journal of General Virology, 1980, vol. 48, pp. 97-104.
Lafon M., Wiktor T.J. Antigenic sites on the ERA rabies virus nucleoprotein and non-structural protein. Journal of General Virology, 1985, vol. 66, pp. 2125-2133. doi:10.1016/0168-1702(87)90023-2.
Dietzschotd B., Lafon M., Wang H., Otvos L. Jr., Celis E., Winner W.H., Koprowski H. Localization and immunological characterization of antigenic domains of the rabies virus internal N and NS proteins. Virus Research, 1987, vol. 8, pp. 103-125.
Minamoto N., Tanaka H., Hishida M., Goto H., Ito H., Naruse S., Yamamoto K., Sugiyama M., Kinjo T., Mannen K., Mifune K. Linear and conformation-dependent antigenic sites on the nucleoprotein of rabies virus. Microbiology and Immunology, 1994, vol. 38, pp. 449-455. doi: 10.1111/j.1348-0421.1994.tb01806.x.
Masatania T., Ito N., Shimizua K., Ito Y., Nakagawa K., Abea M., Yamaoka S., Sugiyama M. Amino acids at positions 273 and 394 in rabies virus nucleoprotein are important for both evasion of host RIG-I-mediated antiviral response and pathogenicity. Virus Research, 2011, vol. 155, pp. 168-174.
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. Journal of General Virology, 1994, vol. 75, pp. 1339-1344.
Katayama S., Yamanaka M., Ota S., Shimizu Y. A new quantitative method for rabies virus by detection of nucleoprotein in virion using Elisa. Journal of Veterinary Medicine Science, 1998, vol. 61, pp. 411-416. doi: 10.1292/jvms.61.411.
Maniatis T., Fritsch E., Sambrook J. Molecularnoe clonirovanie. Metodi geneticheskoi ingenerii [Molecular cloning. A laboratory manual]. Мoscow, Mir, 1982.
Hanahan D. Studies of transformation of Escherichia coli with plasmids. Journal of Molecular Biology, 1983, vol. 166, pp. 557-580. PMID: 6345791.
Sanger F., Nicklen S., Coulson A. DNA sequencing with chain-terminating inhibitors. Proceeding National Academic Science. USA, 1977, vol. 74, pp. 5463-5467. PMID: 271968.
Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences USA, 1979, vol. 76. pp. 4350-4354.
Gato H., Minamoto N., Ito H., Luo T.R., Sugiyama M., Kinjo T., Kavai A. Expression of the nucleoprotein of rabies virus in Escherichia coli and mapping of antigenic sites. Archives of Virology, 1995, vol. 140, pp. 1061-1074. doi: 10.1007/BF01315415.
Fanayi K., Ajorloo M., Mozhgani S.H.R., Irani S., Gholami A. Design, construction and expression of recombinant vector containing the rabies virus nucleoprotein gene. Tehran University Medical Journal, 2014, vol. 72, pp. 294-300.
Yin X., Li Zh., Li J., Yi Y., Zhang Y. et al. Rabies virus nucleoprotein expressed in silkworm pupae at high-levels and evaluation of immune responses in mice. Journal of Biotechnology, 2013, vol. 163, pp. 333-338.
Cao W., He Y., Shi Q., Yang C., Zhang Y. Expression of nucleoprotein gene of CTN strain rabies virus from China in E. coli with antigenicity. Open Journal of Veterinary Medicine, 2013, vol. 3, pp. 309-313. doi: 10.4236/ojvm.2013.37050.
Khassenov B., Sultanculov B.M., Kozhakhmetov S.S., Akhmetov B.S., Ramanculov E.M., Mukanov K.K. Poluchenie recombinantnogo antigena virusa leicoza krupnogo rogatogo scota p24 [Producing recombinant antigen of the virus of bovine leukemia p24]. Biotechnology: theory and practice, 2011, no. 2, pp. 50-56.
Kamzina A.S., Baltabekova A.Zh., Shustov A.V. Virus-like particles presenting conformational epitopes as a promising vaccine platform. Biotechnology: theory and practice, 2012, no. 4, pp. 3-9.
Kamzina A.S., Baltabekova A.Zh., Shustov A.V. Effectivniy metod poluchenia virusopodobnikh chastic, nesuschikh epitopi immunoglobulina cheloveka [Efficient method of producing virus-like particles carrying epitopes of human immunoglobulin]. Biotechnology: theory and practice, 2013, no. 3, pp. 20-25.
Калиева М.Ж., Baltabekova A.Zh., Shustov A.V. Poluchenie i characterizacia recombinantnikh epitopnikh antigenov ischura epizooticheski actualnikh for Kazakhstan serotipov J, A, Asia-1 [Obtaining and characterization of recombinant epitope antigens of the FMD epizootic topical for Kazakhstan serotypes O, A, Asia-1]. Biotechnology: theory and practice, 2014, no. 4, pp. 44-52.
Abeldenov S., Khassenov B. Cloning, expression and purification of recombinant analog of Taq DNA polymerase. Biotechnology. Theory and Practice, 2014, no. 1, pp. 12-16.
Mussakhmetov A., Nurmagambetova A., Abeldenov S., Khassenov B. Purification of recombinant Pfu DNA polymerase by double step affinity chromatography. Biotechnology. Theory and Practice, 2014, no. 2, pp. 42-47.
Abeldenov S., Kirillov S., Kiribayeva A., Silayev D., Khassenov B. Expression, purification and biochemical characterization of recombinant phosphohydrolase AppA in Escherichia coli. Biotechnology. Theory and Practice, 2014, no. 3, pp. 61-65.
Evtikhova E.B., Silaev D.V., Baltabekova A.Zh., Shustov A.V. Termostabilnai recombinantnai phytase proizvedennai v E. coli [Thermostable recombinant phytase produced in E. coli]. Biotechnology: theory and practice, 2014, no. 1, pp. 62-70.
Jiang Y., Luo Y., Michel F., Hogan R.J., He Y., Fu Zh.F. Characterization of conformation-specific monoclonal antibodies against rabies virus nucleoprotein. Archive Virology, 2010, vol. 155, pp. 1187-1192.
Inoue S., Motoi Y., Kashimura T., Ono K., Ya-mada A. Safe and easy monitoring of anti-rabies anti-body in dogs using His-tagged recombinant N-protein. Japanese Journal of Infectious Diseases, 2003, vol. 56, pp. 158-160. PMID: 14583639.