PJB-2022-346
Molecular cloning and prokaryotic expression of the polyprotein gene of tulip breaking virus
Nan Tang, Rongchun Ye and Daocheng Tang
Abstract
The tulips are important bulbous ornamental plants. Tulip breaking virus (TBV) is the most serious virus disease of tulips which is transmitted by aphids. It is very important to pick out the infected bulbs before the aphids start spreading the virus. Notably, a fast and reliable diagnostic approach that can be carried out during the bulb storage period is needed to control the spread of TBV. Since the ELISA kit for the detection of TBV has not been commercialized, the aim of this study was to clone and express TBV protein in E. coli and prepare monoclonal antibodies for the virus. In the current study, the TBV polyprotein gene fragment FK912_gp1 was obtained from TBV infected bulbs of the tulip cultivar ‘Barcelona’ by RT‒PCR. FK912_gp1 was successfully ligated with the expression vector pCold I and transformed into E. coli. The prokaryotic expression vector of pCold I-TBV was constructed. The fusion protein was expressed in E. coli and then purified, at a concentration of 1.24 mg/ml and a purity of greater than 90%. After cell fusion and subcloning, 2 hybridoma cell lines 2B12 and 4G6 were successfully obtained. The results of DAS-ELISA presented that the valence of the obtained monoclonal antibodies reached 512000. These results provide important theoretical support for future studies on the molecular structure, function, and immunogenicity of the TBV polyprotein
To Cite this article:
Tang, N., R. Ye and D. Tang. 2024. Molecular cloning and prokaryotic expression of the polyprotein gene of tulip breaking virus. Pak. J. Bot., 56(2): DOI: http://dx.doi.org/10.30848/PJB2024-2(19)
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