Prokaryotic expression of full coat protein gene of Tomato spotted wilt virus and development of Dot-blot ELISA method for this virus detection
The full coat protein gene of Tomato spotted wilt virus (TSWV) was subcloned into a prokaryotic expression vector pET-32a. Results of restriction enzyme digestion and DNA sequencing show that the insertion direction and sequence were correct and no frameshift mutation existed. This recombinant proka...
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| Format: | Article |
| Language: | English |
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Zhejiang University Press
2008-11-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
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| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2008.06.002 |
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| _version_ | 1850044781505282048 |
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| author | YU Cui DENG Feng-lin YANG Cui-yun WU Jian-xiang |
| author_facet | YU Cui DENG Feng-lin YANG Cui-yun WU Jian-xiang |
| author_sort | YU Cui |
| collection | DOAJ |
| description | The full coat protein gene of Tomato spotted wilt virus (TSWV) was subcloned into a prokaryotic expression vector pET-32a. Results of restriction enzyme digestion and DNA sequencing show that the insertion direction and sequence were correct and no frameshift mutation existed. This recombinant prokaryotic expression vector (pET-32a-TSWV-CP) was used to transform E. coli BL21 (DE3). SDS-PAGE analysis confirmed that the recombinant coat protein with the predicted molecular weight of about 48 kDa was successfully expressed in E. coli BL21 (DE3) strain with the induction of IPTG in the form of a fusion protein. The expressive coat protein was confirmed by 6 × His tag monoclonal antibody and TSWV antiserum raised against TSWV FoPaTs1. The purified recombinant coat protein was used to immunize mouse for production of monoclonal antibody. Using culture supernatants of the hybridoma cell lines, a Dot-blot ELISA method was established for reliable and efficient detection of TSWV in plant samples. |
| format | Article |
| id | doaj-art-90cad0c1012142a3b172e14a8a7df4bc |
| institution | DOAJ |
| issn | 1008-9209 2097-5155 |
| language | English |
| publishDate | 2008-11-01 |
| publisher | Zhejiang University Press |
| record_format | Article |
| series | 浙江大学学报. 农业与生命科学版 |
| spelling | doaj-art-90cad0c1012142a3b172e14a8a7df4bc2025-08-20T02:54:50ZengZhejiang University Press浙江大学学报. 农业与生命科学版1008-92092097-51552008-11-013459760110.3785/j.issn.1008-9209.2008.06.00210089209Prokaryotic expression of full coat protein gene of Tomato spotted wilt virus and development of Dot-blot ELISA method for this virus detectionYU CuiDENG Feng-linYANG Cui-yunWU Jian-xiangThe full coat protein gene of Tomato spotted wilt virus (TSWV) was subcloned into a prokaryotic expression vector pET-32a. Results of restriction enzyme digestion and DNA sequencing show that the insertion direction and sequence were correct and no frameshift mutation existed. This recombinant prokaryotic expression vector (pET-32a-TSWV-CP) was used to transform E. coli BL21 (DE3). SDS-PAGE analysis confirmed that the recombinant coat protein with the predicted molecular weight of about 48 kDa was successfully expressed in E. coli BL21 (DE3) strain with the induction of IPTG in the form of a fusion protein. The expressive coat protein was confirmed by 6 × His tag monoclonal antibody and TSWV antiserum raised against TSWV FoPaTs1. The purified recombinant coat protein was used to immunize mouse for production of monoclonal antibody. Using culture supernatants of the hybridoma cell lines, a Dot-blot ELISA method was established for reliable and efficient detection of TSWV in plant samples.https://www.academax.com/doi/10.3785/j.issn.1008-9209.2008.06.002<italic>Tomato spotted wilt virus</italic>prokaryotic expressionDot-blot ELISA method |
| spellingShingle | YU Cui DENG Feng-lin YANG Cui-yun WU Jian-xiang Prokaryotic expression of full coat protein gene of Tomato spotted wilt virus and development of Dot-blot ELISA method for this virus detection 浙江大学学报. 农业与生命科学版 <italic>Tomato spotted wilt virus</italic> prokaryotic expression Dot-blot ELISA method |
| title | Prokaryotic expression of full coat protein gene of Tomato spotted wilt virus and development of Dot-blot ELISA method for this virus detection |
| title_full | Prokaryotic expression of full coat protein gene of Tomato spotted wilt virus and development of Dot-blot ELISA method for this virus detection |
| title_fullStr | Prokaryotic expression of full coat protein gene of Tomato spotted wilt virus and development of Dot-blot ELISA method for this virus detection |
| title_full_unstemmed | Prokaryotic expression of full coat protein gene of Tomato spotted wilt virus and development of Dot-blot ELISA method for this virus detection |
| title_short | Prokaryotic expression of full coat protein gene of Tomato spotted wilt virus and development of Dot-blot ELISA method for this virus detection |
| title_sort | prokaryotic expression of full coat protein gene of tomato spotted wilt virus and development of dot blot elisa method for this virus detection |
| topic | <italic>Tomato spotted wilt virus</italic> prokaryotic expression Dot-blot ELISA method |
| url | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2008.06.002 |
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