Production Of Monclonal Antibodies Against Tomato Yellow Leaf Curl Virus And Cucumber Green Mottle Mosaic Virus And Their Application

The antibody-based serological method had much success in plant virus detection, because it is sensitive, specific, rapid and simple to conduct, can be conducted on a large-scale. Antibodies of a few plant viruses had been produced in China, but detection of most plant viruses was relied on imported commercial antibodies or ELISA kits. Thus, it is necessary to produce antibodies against important plant viruses, and develop effective serological methods for virus detection, quarantine and prevention. Tomato yellow leaf curl virus (TYLCV) is a species of the family Geminiviridae, causing serious yield losses in tomato production, and cucumber green mottle mosaic virus (CGMMV) is a serious alien invasive pathogen of cucurbit crops. In this study, monoclonal antibodies (MAbs) against TYLCV and CGMMV were producted, and several serological methods were set up for rapid, reliable and sensitive detection.(1) MAbs against TYLCV and their application:The coat protein (CP) gene of TYLCV isolate SH2 (TYLCV-SH2) was amplified by PCR and inserted into a prokaryotic expression vector pET-32a to produce pET-32a-CP, which was used to transform Escherichia coli BL21 (DE3). After induced by IPTG, an about 48 kDa fusion protein was obtained and purified through Ni+ -NTA affinity column. The purified recombinant CP was used to immunize BALB/c mice for producing MAbs. Three hybridoma cell lines secreting MAbs aganint TYLCV were obtained. The results of TAS-ELISA detection showed that the MAb 3E10 could react with four begomoviruses infecting tomato, while the others MAbs (2B2 and 2E3) mainly reacted with TYLCV. A TAS-ELISA method based on MAb 3E10 was set up for TYLCV detection, and this method could successfully detect virus in plant sap at 1:2 560 (w/v, g/mL). The resultes of TAS-ELISA detection of field samples showed begomoviruses were common in tomato plants.(2) MAbs against CGMMV and their application:After selected and cloned, six hybridoma cell lines secreting MAbs against CGMMV were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized by purified CGMMV particles. MAb 5D11 could react strongly with CGMMV, TMV and ORSV, not with ToMV. MAbs 8E3 and 11B12 could react strongly with CGMMV and weakly with TMV and ToMV, while MAbs 4H1,5B10 and 11A4 could react strongly only with CGMMV, but not with the other three tobamoviruses. Based on the most sensitive MAb 4H1, the indirect antigen-coated plate (ACP)-ELISA, Dot-blot ELISA, Tissue-blot ELISA and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) for CGMMV detection were set up. Both ACP-ELISA and Dot-blot ELISA could specifically detect CGMMV in infected cucumber leaf tissue extracts diluted 1:40 960 andl:20 480 (w/v, g/mL), separately. Tissue-blot ELISA is more practical for routine detection of large-scale samples in the field survey. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus and could successfully detect virus in plant sap at 1:102 400 (w/v, g/mL).