Performances and application of antisera produced by recombinant capsid proteins of Cymbidium mosaic virus and Odontoglossum ringspot virus

Abstract

Antisera against important orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), were separately produced using bacterially expressed recombinant capsid proteins (CP), instead of purified virus particles, as immunogens. These antisera were then designated as home-made CymMV CP antiserum (HM-Cy) and home-made ORSV CP antiserum (HM-OR). The high specificity of HM-Cy and HM-OR were confirmed by immunoblot. Their detection limits were determined using indirect-enzyme-linked immunosorbent assay (I-ELISA). Both HM-Cy and HM-OR showed low background reactivity to healthy plants and thus displayed a high S/H ratio (sample OD405/healthy control OD405) in tested orchids. The data indicated that our antisera were efficient and accurate in determination of negative and positive results in ELISA test as commercial antibodies. Therefore, these homemade antisera of CymMV and ORSV are suitable for the certification programme of orchids due to their low cost and high specificity. HM-Cy and HM-OR were further used for a field survey to study the incidence of CymMV and ORSV. The results showed that CymMV is more prevalent than ORSV in Taiwan.

Preparation of Polyclonal Antibodies to Grapevine fanleaf Virus Coat Protein Expressed in Escherichia coli

“…Proteins from several plant viruses have been produced in E. coli and used for raising virus-specific antibodies for immunodiagnosis (Lee and Chang, 2008;Cerovska et al, 2006;Jain et al, 2005;Abou-Jawdah et al, 2004;Cerovska et al, 2003;Hourani and Abou-Jawdah, 2003;Korimbocus et al, 2002;Kumari et al, 2001). In this study, we report the use of GFLV CP expressed in E. coli as the antigen to produce GFLV-specific polyclonal antibodies, which proved to be efficient, when tested in ELISA and Western blotting.…”

Molecular characterization of Indian isolate of peanut mottle virus and immunodiagnosis using bacterial expressed core capsid protein

“…Similar results of expression of recombinant His-tagged CP in the insoluble fraction have been reported for several plant viruses, pelargonium zonate spot virus [27], faba bean necrotic yellows virus [15], sugarcane yellow leaf virus [13], potato mop-top virus [5,6] and groundnut bud necrosis virus [12]. However, for some plant viruses such as sugarcane streak mosaic virus, cucurbit yellow stunting disorder virus, cymbidium mosaic virus, the His-tagged viral CP was expressed in soluble fraction [10,11,16,23]. The PAb against core CP successfully detected the expressed protein as well as the virus in infected peanut leaf samples.…”

Identifikasi Molekuler Broad Bean Wilt Virus 2 (Bbwv2) Dan Cymbidium Mosaic Virus (Cymmv) Asal Tanaman Nilam (Pogostemon Cablin Benth.)

“…Reaksi RT dilakukan selama satu jam pada suhu 42 o C. Amplifikasi cDNA. Untuk amplifikasi DNA, digunakan sepasang primer degenerate untuk BBWV (Kondo et al, 2005) dan satu pasang primer spesifik CymMV (Lee & Chang, 2008) dengan urutan nukletotida yang dapat dilihat pada Tabel 1. cDNA yang dihasilkan dari hasil RT, diamplifikasi dengan teknik PCR pada volume 25 µl yang terdiri atas: air bebas nuklease 9,5 µl, PCR mix (Go Green Taq-Promega) 12,5 µl, primer forward 10 µM dan reverse 10 µM, masing-masing 1 µl dan DNA templat 1 µl. Setiap komponen reaksi dipipet satu persatu, dimasukkan ke dalam tabung eppendorf berukuran 0,5 µl, kemudian dimasukkan dalam mesin PCR.…”

“…degenerate yang disusun dari 3 genom CymMV (AF016914, AY571289, U62963) yang diambil dari GenBank (Lee & Chang, 2008). Ketiga genom CymMV tersebut memiliki homologi yang tinggi dengan isolat CymMV asal Manoko berdasarkan analisa BLAST (Miftakhurohmah et al, 2013).…”