DOI: 10.3724/SP.J.1035.2010.00979

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


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

[1] Menz G, Dolecek C, Schonheit U, et al. Serologic and skin test diagnosis of birch pollen allergy with recombi-nant Bet v 1, the chief allergen of birch [J]. Pneumologic, 1996, 50(9): 632—640

[2] Carrillo T, de Castro F R, Cuevas M, et al. Allergy to limpet [J]. Allergy, 1991, 46(7): 515—519

[3] Chu K H, Wong S H, Leung PSC. Tropomyosin is the major allergen in mollusks: Reverse transcriptase polymerase chain reaction, expression and IgE reactivity [J]. Marine Bio-technology, 2000, 2(5): 499—509

[4] Yunginger J W. Fatal food-induced anaphylaxis [J]. Am. Med. Assoc., 1988, 260(10): 1450—1452

[5] Sampson H A, Mendelson L, Rosen J P. Fatal and near-fatal anaphylactic reactions to food in children and adolescents [J]. N Engl J Med., 1992, 327(6): 380—384

[6] Lü X Z, Liu X M, Yang X G. Preliminary survey on status of food allergy in young Chinese students [J]. Chinese Journal of Food Hygiene, 2005, 17(2): 119—121 [吕相征, 刘秀梅, 杨晓光. 健康人群食物过敏状况的初步调查. 中国食品卫生杂志, 2005, 17(2): 119—121]

[7] Yang Q, Li Q, Yu R H, et al. Studies on the induc-tion of haploid androgenesis in the Japanese scallop Patinoprcten yessoensis by ultraviolet irradiation [J]. Acta Hydrobiologica Sinica, 2006, 3(30): 360—362 [杨青, 李琪, 于瑞海, 等. 虾夷扇贝雄核发育单倍体的人工诱导研究. 水生生物学报, 2006, 3(30): 360—362]

[8] Wild L G, Lehrer S B. Fish and shellfish allergy [J]. Curr Allergy Asthma Rep, 2005, 5(1): 74

[9] Yasushi HASEGAWA. Complete nucleotide sequences of cDNA encoding for tropomyosin isoforms from the catch muscles of scallop Patinopecten yessoensis [J]. Fisheries Sciences, 2001, 67(5): 988—990