Preparation and Application of Polyclonal andMonoclonal Antibodies against the NonstructuralNSs Proteins of Tomato spotted wilt virus andImpatiens necrotic spot virus

• Main Authors: Wei-Ting Tsai, 蔡偉婷
• Other Authors: 葉錫東
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/81896615316690679049

碩士 === 國立中興大學 === 植物病理學系所 === 98 === Members of the Tomato spotted wilt virus (TSWV) serogroup and Impatiens necrotic spot virus (INSV) serotype are regarded as Euro-America type tospoviruses according to their nucleocapsid protein (NP) and geographical distribution. Accurate and prompt detection for members of Euro-America type tospoviruses is crucial for effective quarantine to prevent possible invasion or limit further spreading of these viruses in Taiwan. In this investigation, we attempted to develop polyclonal antibody (antisera) and monoclonal antibodies (MAbs) to INSV and TSWV, two representative and economically important Euro-America type tospoviruses, as effective and accurate detection tools. The tospovirus-encoded nonstructural NSs protein, which accumulates abundantly in infected cells, is the gene silencing suppressor to protect the virus from host gene silencing in plants. In this investigation, the NSs proteins of INSV and TSWV were individually expressed in the bacterial system and the purified proteins were used for producing of antibodies from mouse. The mouse antisera produced against the purified recombinant INSV NSs (INSs) proteins reacted strongly with the crude antigen of INSV and also cross-reacted with three members of the TSWV serogroup tospoviruses, TSWV, Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV), in indirect ELISA and western blot analysis. Similarly, the anti-TSWV NSs (TNSs) antisera reacted with the crude antigens from the three analysed members of TSWV serogroup and INSV serotype. In addition, MAbs against purified NSs proteins were generated by hybridoma technique. The anti-INSs hybridoma line 3E12D4 generated MAb-INSs-3E12D4 that reacted specifically with INSV NSs protein, with a titer (ascitic fluid) up to 2.05 × 106 X. The anti-TNSs hybridoma line 81D9E2 generated MAb-TNSs-81D9E2 that reacted specifically with the NSs protein of TSWV, with titers (ascitic fluids) up to 5.12 × 105 X. Moreover, the MAb-INSs-55G4E7 of anti-INSs hybridoma line 55G4E7 reacted with INSV NSs protein and with the NSs proteins of other two viruses from TSWV serogroup, GRSV and TCSV , with a titer (ascitic fluid) up to 5.12 × 105 X. Hence, the presently produced antisera and MAbs to INSV NSs and TSWV NSs proteins can be used to study the roles of muti-funtion NSs proteins and also as effective tools not only for the specific detection of INSV and TSWV but also for the detection of Euro-America type tospoviruses. In addition, our resilts indicated that the combination of MAb-INSs-55G4E7 and MAb-TNSs-81D9E2 can be used as a two-in-one kit to distinguish the Euro-America type tospoviruses from Asia type tospoviruses, and the three-in-one kit (e.g. MAb-INSs-55G4E7, MAb-TNSs-81D9E2 and the previously developed MAb-WNSs-231E6D12) can be used as a accurate and prompt detection tool for the detection of both Euro-America type and Asia type tospoviruses. A fast and accurate detection method can be established by the cooperation of the specific degenerate primers privously developed in our group with the MAb kits generated in this study.