CLONING,EXPRESSION AND IMMUNOCHARACTERIZATION OF ALLERGEN TROPOMYOSIN IN CRAB

Allergic disease is regarded as one of three diseases that need prevention and cure in the 21st century.Food hypersensitivity is a normal allergic disease,which can provokes dizziness,headache,chest distress,itch of skin and so on,so it is necessary to study the food allergens,especially seafood allergens.This study was undertaken to clone,express and immunocharacterize the allergen tropomyosin from crab(Portunus sanguinolentus).Degenerate primers were designed according to the conserved sequence of tropomyosin by means of bioinformatics and molecular biological methods.The RT-PCR was applied to clone the full-length allergen genes from crab and the sequences were analyzed.The specific primers were designed.The ORF of tropomyosin of crab was subcloned into the expression vector pET-28a.Expression of the recombinant crab 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.IgE reactivity of recombinant crab tropomyosin was investigated by Western-blot.After SDS-PAGE,the purified recombinant crab tropomyosin was digested by trypsin and analyzed by MALDI-TOF-MS.A novel full-length cDNA clone was obtained,which includes an open reading frame coding for 285 amino acids.The predicted amino acid sequences of the tropomyosin showed that its pI was approximately 4.71 and theoretical molecular weight was approximately 32.8kD.Sequence analysis showed that this gene shared high identities(80%)with allergen tropomyosin from many crustaceans and other invertebrates,such as 97%,92% and 83% identity with Chaf 1(Q9N2R3) from Charybdis feriatus,Mete 1(Q25456) from Metapenaeus ensis and Pera 7(Q9UB83) from Periplaneta Americana,respectively.The deduced protein was therefore regarded as crab allergen and named as Pors 1(EMBL/GenBank database entry No.EF143836).After over expressed in Escherichia coli BL21(DE3),the recombinant protein was purified through affinity chromatography with Ni2+ coupled to sepharose.Western-blot analysis showed that the recombinant allergen had IgE-reactivity with sera from crab allergic patients.The result of MALDI-TOF-MS indicated that this recombinant allergen was indeed the crab tropomyosin.In short,the crab tropomyosin was cloned and expressed successfully in BL21(DE3),which had reactivity with sIgE.All of these studies will be used as a base for further study on structure and function of crab allergens,also for the specific diagnosis and immunotherapy in crab hypersensitivity disease.