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) Department of Biology, 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) Department of Agronomy, Faculty of Agriculture, Universitas Gadjah Mada, Jalan Flora, Bulaksumur, Yogyakarta 55281, Indonesia
(5) Department of Biology, Faculty of Science and Technology, Airlangga University, C Campus, Jalan Mulyorejo, Surabaya 60115, Indonesia
(6) Department of Biology, Faculty of Science and Technology, Airlangga University, C Campus, Jalan Mulyorejo, Surabaya 60115, Indonesia
(*) Corresponding Author

Abstract


Sugarcane mosaic virus (SCMV, genus Potyvirus, familia 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 production countries, researches on the definement of SCMV from Indonesian isolate based on molecular study are very limited. This study aimed to obtain a proper recombinant antigens emanating from coat protein of SCMV from Indonesian isolate for polyclonal antibody production that can be used for immunodiagnosis purposes. A gene encodes the coat protein of SCMV (CP-SCMV) was amplified by the reverse transcription polymerase chain reaction and cloned into vector pJET1.2. The cDNA was inserted into 6X His-tag expression plasmid of pET28a(+) and overexpressed in Escherichia coli BL21(DE3) to produce recombinant protein. The highest expression had performed on 0.1M IPTG induction media for 5 hours at 37oC. SDS-PAGE analysis clarified that recombinant CP-SCMV remains as an insoluble fraction. The purifications were performed by the affinity Ni-NTA resin which followed by electroelution to obtain highly purified proteins. To meet quality requirements of a proper antigen the highly purified protein was concentrated. Molecular weight of rCP-SCMV (approximately 40 kDa) was clearly observed by 10% SDS-PAGE at the concentration of 16.184 mg/mL. 


Keywords


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

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

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