doi:

DOI: 10.3724/SP.J.1035.2010.00979

Acta Hydrobiologica Sinica (水生生物学报) 2010/34:5 PP.979-983

CLONING, EXPRESSION AND PURIFICATION OF ALLERGEN TROPOMYOSIN FROM PATINOPECTEN YESSOENSIS AND IDENTIFICATION OF ITS ALLERGIC ACTIVITY


Abstract:
This study was undertaken to clone, express and immunocharacterize the allergen tropomyosin from Patinopecten yessoensis. Bioinformatic method was used for the comparative analysis of numerous homologous animal food tropomyosin sequences. Conservative domains among the sequences were determined for degenerate primer designing. The RT- PCR was applied to clone the full- length allergen genes from Patinopecten yessoensis and the se-quences were analyzed. The specific primers were designed. The complete coding cDNA sequence including the start and the stop codons of tropomyosin of Patinopecten yessoensis was subcloned into the expression vector pET 28a. Expression of the recombinant Patinopecten yessoensis tropomyosin was carried out in Escherichia coli BL21 (DE3) and the purification of the recombinant protein was performed via affinity chromatography with Ni2+ coupled to sepharose. Protein from E. coli lysate and purified recombinant tropomyosin were analyzed by SDS-PAGE. IgE reactivity of recombinant Patinopecten yessoensis tropomyosin was investigated by Western-blotting. The cloned cDNA ORF sequence contained 855 bp and encoded 284 amino acids. The GenBank accession number of the clones was EU839640. Sequence analysis showed that this clone shared high identities with tropomyosin from Patinopecten yessoensis. Nucleotide and amino acid comparison showed that this protein was the Patinopecten yessoensis tropomyosin. The recombinant allergen tropomyosin was highly expressed in E. coli BL21 (DE3) with the molecular weight of about 36 kD under induction of IPTG and purified by 6-His-tag purification system. And the recombinant allergen was identified as its affinity to IgE antibodies from the scallop patient sera by Western blotting method. Immunoassay showed that the recombinant allergen has good IgE binding capacity. We obtained recombinant Patinopecten yessoensis tropomyosin with good allergenicity in this study, which would be used as a base for further study on Patinopecten yessoensis related allergy.

Key words:Patinopecten yessoensis,Allergen,Tropomyosin,Cloning and expression

ReleaseDate:2014-07-21 15:25:22



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