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

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


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 37oC. 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. 


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

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