Royalisin is an antibacterial peptide found in Royal Jelly. Two gene fragments of Chinese honeybee (Apis cerana cerana) head, 280 bp cDNA encoding pre-pro-Acc-royalisin (PPAR) of 95 amino acid residues, and 165 bp cDNA encoding mature Acc-royalisin (MAR) of 51 amino acid residues were cloned into the pGEX-4T-2 vector. They were then transformed individually into Escherichia coli for expression. Two expressed fusion proteins, glutathione S-transferase (GST)-PPAR of 36 kDa and GST-MAR of 32 kDa were obtained, which were cross reacted with GST antibody accounting for up to 16.3% and 15.4% of bacterial protein, respectively. In addition, 41% of GST-PPAR and nearly 100% of GST-MAR were soluble proteins. Both lysates of the two purified fusion proteins displayed antibacterial activities, similar to that of nisin against Gram-positive bacteria strains, Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus. MAR peptide released from the thrombin-cleaved GST-MAR fusion protein has a stronger antibacterial activity than that of GST-MAR fusion protein.

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Abstract:Bee venom phospholipase A 2 (BvPLA 2 ) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. In this work, a new BvPLA 2 (AccPLA 2 ) gene from the Chinese honeybee (Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector. Tn-5B-4 (Tn) cells were transfected with the recombinant bacmid DNA for expression. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa. Products of hexahistidine AccPLA 2 fusion protein accumulated up to 5.32% of the total cellular proteins. The AccPLA 2 fusion protein was cross reactive with the anti-AmPLA 2 (BvPLA 2 of the European honeybee, Apis mellifera) polyclonal serum. The reaction resulted in a double glycosylation band, which agrees with the band generated by the native AmPLA 2 in Western blot analysis. The PLA 2 activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 μmol/(min·mg). In summary, the recombinant AccPLA 2 protein, a native BvPLA 2 -like structure with corresponding biological activities, can be glycosylated in Tn cells. These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA 2 in the pharmaceutical industry.