Zhong yao cai = Zhongyaocai = Journal of Chinese medicinal materials · 2011
Prokaryotic expression of CP gene of Fritillary virus Y infecting Thunberg fritillary and antiserum preparation.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Wei Chuan-Bao, Wei Yang-Yang, Yang Yu, Liu Shi-Liang, Hu Hao-Yu, He Yue
Аннотация
OBJECTIVE: To prepare antiserum against Fritillary virus Y (FVY) CP for detecting FVY and study serological relationships with other viruses.
METHODS: Specific primer was designed according to Genbank (accession: AM039800) to amplify CP gene of FVY infecting Thunberg fritillary. Sequence relationship with other potyviruses was made by Blast. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys E strain. The object protein was purified by 12% SDS-PAGE firstly and subsequently 5% - 20% gradient SDS-PAGE. The antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis. The reactivity of the antiserum produced to FVY CP was tested by Western blot against the over-expressed coat proteins of 17 potyviruses. The ability to combine with nature FVY particles was confirmed by ELISA analysis.
RESULTS: It shared 81.2% nucleotide acids identities with TrVY (Tricyrtis virus Y, AY 864850) CP gene, 68.1% with SMV-P (Soybean mosaic virus Pinellia strain, AJ507388. 2) CP gene and 67.2% with ZYMV (Zucchini yellow mosaic virus Luan isolate) CP gene. The prepared antiserum was special to FVY CP, also reacted moderately to the expressed CP of SMV-P (Soybean mosaic virus Pinellia strain) and weakly to that of ZYMV (Zucchini yellow mosaic virus Luan isolate).
CONCLUSION: The antibody could combine to nature FVY particles and the antiserum is suitable for FVY detection by ELISA in large scale.
METHODS: Specific primer was designed according to Genbank (accession: AM039800) to amplify CP gene of FVY infecting Thunberg fritillary. Sequence relationship with other potyviruses was made by Blast. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys E strain. The object protein was purified by 12% SDS-PAGE firstly and subsequently 5% - 20% gradient SDS-PAGE. The antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis. The reactivity of the antiserum produced to FVY CP was tested by Western blot against the over-expressed coat proteins of 17 potyviruses. The ability to combine with nature FVY particles was confirmed by ELISA analysis.
RESULTS: It shared 81.2% nucleotide acids identities with TrVY (Tricyrtis virus Y, AY 864850) CP gene, 68.1% with SMV-P (Soybean mosaic virus Pinellia strain, AJ507388. 2) CP gene and 67.2% with ZYMV (Zucchini yellow mosaic virus Luan isolate) CP gene. The prepared antiserum was special to FVY CP, also reacted moderately to the expressed CP of SMV-P (Soybean mosaic virus Pinellia strain) and weakly to that of ZYMV (Zucchini yellow mosaic virus Luan isolate).
CONCLUSION: The antibody could combine to nature FVY particles and the antiserum is suitable for FVY detection by ELISA in large scale.
Предметные рубрики MeSH
Amino Acid SequenceAnimalsBase SequenceBlotting, WesternCapsid ProteinsChinaCloning, MolecularDNA PrimersEnzyme-Linked Immunosorbent AssayEscherichia coliFritillariaGene ExpressionImmune SeraMiceMolecular Sequence DataPhylogenyPinelliaPolymerase Chain ReactionPotyvirusRecombinant ProteinsGlycine max