Cloning, expression and purification of pan-allergen tropomyosin from Squilla oratoria and identification of its allergic activity

ObjectiveTo clone the tropomyosin gene from squilla oratories and produce its recombinant protein and investigate its allergenicity.MethodsThe cDNA of tropom yosin was cloned using specific priners from the total RNA of squilla oratories The cloned gene was inserted into pMD 18-T vector and digested by Nde I and Hind Ⅲ The cDNA was sequenced and then subcloned into pET-28a expression vector The cloned tropom yosin cDNA gene was expressed in Escherichia coliBL21 (DE3) by IPTG induction.The recombinant tropomyosin was purified by metal (Ni²⁺)chelating affinity chromatography.The allergenicity of the recombinant tropomyosin was examfined by western blotting method.ResultsThe cloned cDNA ORF sequence contained 855 by and encoded 284 aminoacids (GenBank database entry Na EF584510).The recombinant allergen tropomyosinwas highly expressed in E.coli BL21 (DE3) with the molecular weight of about 36 kDa under induction of IPTG and purified by 6-His-tag purification system.And the recombinant allergen was identified as its affinny to IgE antibodies from the shrinp-allergic patient sera.ConclusionRecombinant squilla oratories tropomyos in with good allergenicity was obtained in this study.