Expression and purification of recombinant coat protein of sugarcane mosaic virus from Indonesian isolate as an antigen for antibody production

Natalia Tri Astuti; Nurmalasari Darsono; Suvia Widyaningrum; Widhi Dyah Sawitri; Sri Puji Astuti; Win Darmanto

doi:10.22146/ijbiotech.45551

Expression and purification of recombinant coat protein of sugarcane mosaic virus from Indonesian isolate as an antigen for antibody production

https://doi.org/10.22146/ijbiotech.45551

Natalia Tri Astuti⁽¹⁾, Nurmalasari Darsono⁽²⁾, Suvia Widyaningrum⁽³⁾, Widhi Dyah Sawitri⁽⁴⁾, Sri Puji Astuti⁽⁵⁾, Win Darmanto^(6*)

(1) Biotechnology Laboratory, PT. Perkebunan Nusantara XI, Jalan Merak 1, Surabaya 60175, Indonesia
(2) Biology Department, Faculty of Science and Technology, Airlangga University, C Campus, Jalan Mulyorejo, Surabaya 60115, Indonesia
(3) Post Graduate Program for Biotechnology, University of Jember, Jalan Kalimantan No. 37, Jember 68121, Indonesia
(4) Agronomy Department, Faculty of Agriculture, Universitas Gadjah Mada, Jalan Flora, Bulaksumur, Yogyakarta 55281, Indonesia
(5) Biology Department, Faculty of Science and Technology, Airlangga University, C Campus, Jalan Mulyorejo, Surabaya 60115, Indonesia
(6) Biology Department, Faculty of Science and Technology, Airlangga University, C Campus, Jalan Mulyorejo, Surabaya 60115, Indonesia
(*) Corresponding Author

Abstract

Sugarcane mosaic virus (SCMV, genus Potyvirus, family Potyviridae) is a prominent pathogen of sugarcane (Saccharum sp. hybrids). It can cause losses in susceptible varieties, in crop as well as sugar production, economically. Although it has been studied in major sugar-producing countries, research on the definement of SCMV from Indonesian isolates based on molecular study has been very limited. This study aimed to obtain a proper recombinant antigens emanating from coat protein of SCMV from Indonesian isolate in order to produce polyclonal antibodies that cann be used for immunodiagnosis assays in a subsequent study. A gene-encoding coat protein of SCMV (CP-SCMV) was amplified using RT-PCR and cloned into vector pJET1.2. The cDNA was inserted into 6X His-tag expression plasmid of pET28a(+) and over-expressed in Escherichia coli BL21(DE3) to produce a recombinant protein. The highest expression was found in 0.1M IPTG induction media for 5 h at 37^oC. SDS-PAGE analysis clarified that the recombinant CP-SCMV remained as an insoluble fraction. Purifications was carried out by the affinity Ni-NTA resin, followed by electroelution to obtain a highly purified protein. To meet the quality requirements of a proper antigen, the highly purified protein was concentrated. A molecular weight of the rCP-SCMV (approximately 40 kDa) was clearly observed by 10% SDS-PAGE at the concentration of 16.184 mg/mL.

Keywords

antigen; cDNA; recombinant coat protein; sugarcane mosaic virus (SCMV)

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References

Abdel-Salam AM, El-Attar AK, Gambley CF. 2014. Production of polyclonal antisera to a recombinant coat protein of Potato virus Y expressed in Escherichia coli and its application for immunodiagnosis. International Journal of Virology. 10(1):1–16. doi:10.3923/ijv.2014.1.16.

Abdel-Salam AM, El-Attar AK, Soliman AM. 2013. The use of native and denatured recombinant coat protein forms for induction of good quality antisera for Potato virus X and Potato leaf roll virus. American Journal of Research Communication. 1(17):70–86.

Addy H, Nurmalasari, Wahyudi A, Sholeh A, Anugrah C, Iriyanto F, Darmanto W, Sugiharto B. 2017. Detection and response of sugarcane against the infection of Sugarcane mosaic virus (SCMV) in Indonesia. Agronomy. 7(3):50. doi:10.3390/agronomy7030050.

Akbari V, Sadeghi HMM, Jafarian-Dehkordi A, Chou CP, Abedi D. 2015. Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology. Res Pharm Sci. 10(1):75–83.

Anwar S. 2005. Impact of sugarcane disease on cane and sugar yield, in proceedings of the 3rd agricultural biotechnology workshop. Agricultural Biotechnology Research Institute, Faisalabad, Pakistan.

Barrell PJ, Liew OW, Conner AJ. 2004. Expressing an antibacterial protein in bacteria for raising antibodies. Protein Expression and Purification. 33(1):153–159. doi:10.1016/j.pep.2003.08.026.

Borowiec M, Gorzkiewicz M, Grzesik J, Walczak-Drzewiecka A, Salkowska A, Rodakowska E, Steczkiewicz K, Rychlewski L, Dastych J, Ginalski K. 2016. Towards engineering novel PE-Based immunotoxins by targeting them to the nucleus. Toxins. 8(11):321. doi:10.3390/toxins8110321.

Carson M, Johnson DH, McDonald H, Brouillette C, DeLucas LJ. 2007. His-tag impact on structure. Acta Crystallographica Section D Biological Crystallography. 63(3):295–301. doi:10.1107/S0907444906052024.

Crescenzi A, d’Aquino L, Nuzzaci M, Ostuni A, Bavoso A, Comes S, De Stradis A, Piazzolla P. 1997. Production of strain specific antibodies against a synthetic polypeptide corresponding to the N-terminal region of the Plum pox potyvirus coat protein. Journal of Virological Methods. 69(1–2):181–189. doi:10.1016/s0166-0934(97)00158-4.

Damayanti TA, Putra LK. 2011. First occurrence of Sugarcane streak mosaic virus infecting sugarcane in Indonesia. Journal of General Plant Pathology. 77(1):72–74. doi:10.1007/s10327-010-0285-7.

Fajardo TVM, Barros DR, Nickel O, Kuhn GB, Zerbini FM. 2007. Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies. Fitopatologia Brasileira. 32(6):496–500. doi:10.1590/S0100-41582007000600007.

Gay G, Wagner DT, Keatinge-Clay AT, Gay DC. 2014. Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae. Plasmid. 76:66–71. doi:10.1016/j.plasmid.2014.09.005.

Gulati-Sakhuja A, Sears JL, Nuñez A, Liu H-Y. 2009. Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis. Journal of Virological Methods. 160(1–2):29–37. doi:10.1016/j.jviromet.2009.04.005.

Haider MS, Afghan S, Riaz H, Tahir M, Javed MA, Rashid N, Iqbal J. 2011. Identification of two Sugarcane mosaic virus (SCMV) variants from naturally infected sugarcane crop In Pakistan. Pak J Bot. 43(2):1157–1162.

Hema M, Kirthi N, Sreenivasulu P, Savithri HS. 2003. Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of Sugarcane streak mosaic virus isolates from Southern India. Archives of Virology. 148(6):1185–1193. doi:10.1007/s00705-003-0015-y.

Hoseini S, Sauer MG. 2015. Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering. Journal of Biological Engineering. 9(1):2. doi:10.1186/1754-1611-9-2.

Institutional Animal Care and Use Committee. 2016. Administering complete freund’s adjuvant (CFA) and other adjuvants. Atlanta: Emory University.

Kaur U, Khurana S, Saikia UN, Dubey ML. 2013. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis. Experimental Parasitology. 135(3):486–496. doi:10.1016/j.exppara.2013.08.011.

Khalil SM, Salama MI, Abdel-Hamid IA, Nour El-Din HA, Sadik AS. 2007. Effective detection of Banana bunchy top nanovirus using DNA probes. Pak J Biotechnol. 4(1–2):113–118.

Khan S, Jan AT, Mandal B, Haq QMohdR. 2012. Immunodiagnostics of Cucumber mosaic virus using antisera developed against recombinant coat protein. Archives Of Phytopathology And Plant Protection. 45(5):561–569. doi:10.1080/03235408.2011.588043.

Khatabi B, He B, Hajimorad MR. 2012. Diagnostic potential of polyclonal antibodies gainst bacterially expressed recombinant coat protein of Alfalfa mosaic virus. Plant Disease. 96(9):1352–1357. doi:10.1094/PDIS-08-11-0683-RE.

Koohapitagtam M, Nualsri C. 2013. Production of polyclonal antibodies speciﬁc to the recombinant coat protein of Blackeye cowpea mosaic virus and Its use in disease detection. Kasetsart Journal: Natural Science. 47:603–613.

Leenaars M, Hendriksen CFM. 2005. Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. ILAR Journal. 46(3):269–279. doi:10.1093/ilar.46.3.269.

Luo Y, Glisson JR, Jackwood MW, Hancock RE, Bains M, Cheng IH, Wang C. 1997. Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73. Journal of Bacteriology. 179(24):7856–7864. doi:10.1128/jb.179.24.7856-7864.1997.

Mohammadi MR, Hajieghrari B. 2009. Sugarcane mosaic virus: The causal agent of mosaic disease on sorghum (Sorghum bicolor L.) in Tehran province of Iran. African Journal of Biotechnology. 8(20):5271–5274.

Ohhashi T, Moritani C, Andoh H, Satoh S, Ohmori S, Lottspeich F, Ikeda M. 1991. Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate—polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies. Journal of Chromatography A. 585(1):153–159. doi:10.1016/0021-9673(91)85069-R.

Rimler RB. 2001. Purification of a cross-protective antigen from Pasteurella multocida grown in vitro and in vivo. Avian Diseases. 45(3):572–580. doi:10.2307/1592897.

Sambrook J, Russell DW. 2001. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press.

Saraswat M, Musante L, Ravidá A, Shortt B, Byrne B, Holthofer H. 2013. Preparative purification of recombinant proteins: current status and future trends. BioMed Research International. 2013:1–18. doi:10.1155/2013/312709.

Shukla DD, Frenkel MJ, McKern NM, Ward CW, Jilka J, Tosic M, Ford RE. 1992. Present status of the sugarcane mosaic subgroup of potyviruses. In: Barnett OW, editor. Potyvirus Taxonomy. Vol. 5. Vienna: Springer Vienna. p. 363–373.

Sreenivasulu M, Gopal DVRS. 2010. Development of recombinant coat protein antibody based IC-RT-PCR and comparison of its sensitivity with other immunoassays for the detection of Papaya ringspot virus isolates from India. The Plant Pathology Journal. 26(1):25–31. doi:10.5423/PPJ.2010.26.1.025.

Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. 1990. [6] Use of T7 RNA polymerase to direct expression of cloned genes. In: Methods in Enzymology. Vol. 185. Elsevier. p. 60–89. https://linkinghub.elsevier.com/retrieve/pii/007668799085008C.

Thanasarasakulpong A, Poolperm P, Tangjitjaroen W, Varinrak T, Sawada T, Pfeiffer D, Sthitmatee N. 2016. Comparison of the effect of two purification methods on the immunogenicity of recombinant outer membrane protein H of Pasteurella multocida Serovar A:1. Veterinary Medicine International. 2016:1–7. doi:10.1155/2016/2579345.

Vázquez-Iglesias L, Estefanell-Ucha B, Barcia-Castro L, Páez de la Cadena M, Álvarez-Chaver P, Ayude-Vázquez D, Rodríguez-Berrocal FJ. 2017. A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum. PeerJ. 5:e3407. doi:10.7717/peerj.3407.

Xu D-L, Park J-W, Mirkov TE, Zhou G-H. 2008. Viruses causing mosaic disease in sugarcane and their genetic diversity in southern China. Archives of Virology. 153(6):1031–1039. doi:10.1007/s00705-008-0072-3.

DOI: https://doi.org/10.22146/ijbiotech.45551