RESUMO
Spiroplasma eriocheiris caused massive mortality of Chinese mitten crab Eriocheir sinensis but little is known about the molecular characteristics of this microorganism. We described here the identification of a spiralin-like protein (SLP31) from S. eriocheiris and expression in Escherichia coli. Analysis of the nucleotide sequence revealed that the clone has an open reading frame of 837 bp encoding a protein of 279 amino acids. Theoretical isoelectric point and molar mass for SLP31 are 7.72 and 31 kDa, respectively. The similarity of SLP31 deduced amino acid sequence shared with the spiralin from other species indicated that the gene may be a member of spiralin family. The TGA codon in Spiroplasma serves not as a stop signal but as a code for the amino acid tryptophan. After cloning the SLP31, the gene was site-mutated from TGA to TGG and transcribed in E. coli to full expression of SLP31. The purified recombinant protein was used to determine the immune reactivity by Western blotting which suggests that SLP31 could be a good antigen for immunodiagnostic of tremor disease in E. sinensis.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Técnicas Bacteriológicas/métodos , Braquiúros/microbiologia , Spiroplasma/isolamento & purificação , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Sequência de Bases , Western Blotting , China , Clonagem Molecular , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Imunoensaio/métodos , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNA , Homologia de Sequência , Spiroplasma/genética , Spiroplasma/imunologiaRESUMO
Spiroplasma eriocheiris causes tremor disease (TD) of Chinese mitten crab Eriocheir sinensis, but little is known about the molecular characterization of this pathogen. The present study was undertaken to identify a unique gene of spiroplasma, spiralin-like protein (SLP25) and analyze its character. The full-length DNA of SLP25 is 699 bp encodes a protein of 232 amino acid residues. The theoretical isoelectric point and molecular weight for the SLP25 are 4.50 and 24.67 kDa, respectively. The similarity of SLP25 deduced amino acid sequence, shared with the spiralin from other species, indicated that the gene might be a member of the spiralin family. After cloning the SLP25, the gene was site-mutated from TGA to TGG and transcribed in E. coli to full expression of the recombinant SLP25. Anti-SLP25 serum was prepared by immunization of rabbits. The titer of anti-SLP25 serum was about 1:20000. A SLP25 band was detected by anti-SLP25 antibody using the total proteins of S. eriocheiris. A specific immunoreactive band was detected when anti-S. eriocheiris serum was opposed to the recombinant protein. This finding suggests that SLP25 could be a good antigen for immunodiagnosis of TD of E. sinensis.
