Molecular characterization and antibody preparation of a Turnip mosaic virus isolate infecting calla lily
Date Issued
2004
Date
2004
Author(s)
Wu, Chun-I
DOI
zh-TW
Abstract
A calla lily leaf sample infected by several viruses was collected from the field in 2002. When Chenopodium quinoa was inoculated by the leaf extract, it appeared chlorotic local lesions. After three successive single lesion isolation, a virus isolate was obtained. The yellow spot and stripe symptoms showed when the isolate was transferred back to tissue-cultured transplants of calla lily. The virus isolate reacted positively with the monoclonal antibody specific against potyvirus group by indirect enzyme-linked immunosorbent assay (ELISA). Flexuous virus particles about 750 nm in length were observed by the transmission electron microscopy. All of above evidences revealed that the isolate is a potyvirus but different from those calla lily-infected potyviruses studied in our laboratory, including Dasheen mosaic virus (DsMV), Zantedeschia mosaic virus (ZaMV) and Zantedeschia mild mosaic virus (ZaMMV). To further characterize the viral molecular nature, a pair of degenerate primers designed according to the conserved sequences of the reported potyviruses was used to amplify 1.7 kb fragment by RT-PCR. After cloning and sequencing, viral genomic sequences from partial NIb gene to 3’UTR were obtained and they are similar to Turnip mosaic virus (TuMV) by the database alignment. Therefore, this virus isolate was named as TuMV-ZAN. Using the same clonging strategy, we obtained the full length genomic sequences of TuMV-ZAN including 5’ and 3’ UTR, P1, HC-Pro, P3, 6K1, CI, 6K2, NIa, NIb and CP genes. Another TuMV isolate infecting calla lily was found and sequenced in 2004, but its nucleotide sequences of CP was only 89％ identity with those of TuMV-ZAN. This isolate was named TuMV-ZAN2. Phylogenetic analysis of the CP gene indicates that the ZAN and ZAN2 isolates are located at world-B and basal-BR group, respectively.
In order to obtain the antiserum of TuMV for further study and field survey, we cloned the CP gene of TuMV-ZAN into pET-29a(+) expression vector. After transformation to Escherichia coli BL21 (DE3) and induction by IPTG, the recombinant CP of TuMV-ZAN was expressed and purified to immunize rabbits. The titer and specificity of the prepared TuMV antiserum was tested by indirect-ELISA and western blot analysis. Besides, we used the same kind of expressed CP to immunize mouse and obtained a hybridoma cell line－Mab403 after screening. We found that the monoclonal antibody (Mab403) could reacte not only with TuMV but with at least eight other potyviruses.
Subjects
彩色海芋
單元抗體
蕪菁嵌紋病毒
monoclonal antibody
Zantedeschia
Turnip mosaic virus
calla lily
Type
other
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