Objective: To clone and express the arginine kinase (AK) gene of Blattella germanica and analyze its immune activity.
Methods: The cDNA of AK was cloned using specific primers from the total RNA of Blattella germanica The open reading frame (ORF) of AK was cloned into pET-28A vector, and expressed in Escherichia coli BL21(DE3) with IPTG induction. The recombinant protein was purified by Ni2+ chelating affinity chromatography. The recombinant protein was detected by SDS-PAGE, and its immune activity was analyzed by Western blotting.
Results: The cloned cDNA ORF sequence (GenBank accession No. FJ514482) contained 1071bp and encoded 356 amino acids. Its sequence homology with the published one (GenBank accession No. EU429466) was 97.2% at nucleotide level. The recombinant containing recombinant plasmid pET-28a-AK expressed a soluble protein of AK (Mr 45 000) after being induced with IPTG. The recombinant AK protein was recognized by sera of allergic patients, indicating that the recombinant AK protein has an adequate response activity.
Conclusion: The AK gene of Blattella germanica has been cloned and the recombinant AK protein has been confirmed with immune activity.