Cloning, expression and purification of arginine kinase from Locusta migratoria manilensis and its allergic activity
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Abstract： This investigation aimed to clone the Locusta migratoria manilensis arginine kinase (AK) gene，produce its recombinant protein and investigate the protein’s allergenicity. The cDNA of AK was cloned, using specific primers, from the total RNA of L. m. manilensis. The cloned gene was inserted into pMD18-T vector and digested by EcoR I and Xho I. The cDNA was sequenced and subcloned into a pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli Rosetta by IPTG induction. The recombinant AK (rAK) was purified by metal (Ni2+) chelating affinity chromatography. Its allergenicity was examined by Western blotting. The cloned cDNA ORF sequence contained 1 068 bp and encoded 355 amino acids. Its sequence homology with the published sequence (Accession no. DQ513322) was 98% at nucleotide level. The allergen rAK was highly expressed in E. coli as a soluble protein with a molecular weight of about Mr 40 000 under induction with IPTG and purified by a 6Histag purification system. Under both nondenaturalization and denaturalization conditions, the recombinant allergen was identified by its affinity to IgE antibodies from the cockroachallergic patient sera by Western blotting. It is concluded that recombinant arginine kinase with proper allergenicity was successfully obtained.