Letters in applied microbiology · 2010
Identification and characterization of Japanese encephalitis virus envelope protein gene from swine.
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Fan J-M, Luo J, Zhang G-P, Chen L, Teng M, Yang M-F, Wang L, Wang C-Q
Аннотация
AIMS: Identification and characterization of Japanese encephalitis virus (JEV) envelope protein gene from swine.
METHODS AND RESULTS: Genomic RNA was separated from JEV isolated strain Henan-09-03, and used as templates for cDNA synthesis of E gene. The cDNA of E gene was amplified by RT-PCR and cloned into the pMD19-T-Vector and confirmed by sequencing. The cloned gene was then subcloned into the pET-32a and was introduced into Escherichia coli BL21 (DE3) for expression. The E protein was purified by Ni chelating column-based affinity chromatography. The molecular weight of expressed protein was about 50 kDa. Compared with the published sequence of SA14 (AF495589), the homology of the nucleotide sequence was 98% and the seven mutations resulting in amino acid substitutions at Leu 36 Ser, Leu107 Val, Ala167 Thr, Asn 230 Ser, Leu 340 Pro, Asn 430 Ile, Phe 448 Leu. Phylogenetic analysis of the E sequence of isolated strain classified it within genotype III of the JEV. The result of Western blotting indicated that the antigenicity of the protein was specific.
CONCLUSIONS: The stable expression of the protein and the analysis of its antigenic specificity provide the foundation for developing the ELISA early stage diagnosis kit.
SIGNIFICANCE AND IMPACT OF THE STUDY: As coating antigen, the recombinant E protein served a good source in the indirect ELISA method for the detection of JEV antibody.
METHODS AND RESULTS: Genomic RNA was separated from JEV isolated strain Henan-09-03, and used as templates for cDNA synthesis of E gene. The cDNA of E gene was amplified by RT-PCR and cloned into the pMD19-T-Vector and confirmed by sequencing. The cloned gene was then subcloned into the pET-32a and was introduced into Escherichia coli BL21 (DE3) for expression. The E protein was purified by Ni chelating column-based affinity chromatography. The molecular weight of expressed protein was about 50 kDa. Compared with the published sequence of SA14 (AF495589), the homology of the nucleotide sequence was 98% and the seven mutations resulting in amino acid substitutions at Leu 36 Ser, Leu107 Val, Ala167 Thr, Asn 230 Ser, Leu 340 Pro, Asn 430 Ile, Phe 448 Leu. Phylogenetic analysis of the E sequence of isolated strain classified it within genotype III of the JEV. The result of Western blotting indicated that the antigenicity of the protein was specific.
CONCLUSIONS: The stable expression of the protein and the analysis of its antigenic specificity provide the foundation for developing the ELISA early stage diagnosis kit.
SIGNIFICANCE AND IMPACT OF THE STUDY: As coating antigen, the recombinant E protein served a good source in the indirect ELISA method for the detection of JEV antibody.
Предметные рубрики MeSH
Amino Acid SubstitutionAnimalsBlotting, WesternChromatography, AffinityCloning, MolecularCluster AnalysisEncephalitis Virus, JapaneseEscherichia coliGene ExpressionHumansMolecular WeightPhylogenyRNA, ViralRecombinant ProteinsReverse Transcriptase Polymerase Chain ReactionSequence Homology, Nucleic AcidSwineViral Envelope Proteins