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.

Three clones of AccMRJP1 were screened out from the sequenced brain cDNA library of Chinese honeybee according to the expression sequence tag(EST) of AccMRJP1.Through identifying with polymerase chain reaction(PCR) and sequencing,a AccMRJP1 clone containing an open reading frame(ORF) of 1302 nucleotides encoding a protein of 433 amino acids was determined.The AccMRJP1 had 99.8% similarity with the previously reported AccMRJP1 sequence(AY279539) in amino acid sequences.The AccMRJP1 was sub-cloned into the prokaryotic expression vector pGEX-4T-2 for fusion expression in Escherichia coli BL21.Analysis result of the SDS-PAGE showed that the expression product contained a specific band of protein about 76 ku in size and accumulated up to about 17.7% of the total bacterial proteins.The fusion protein was cross reactive with glutathione S-transferase(GST) polyclonal antibody and was purified through affinity chromatography,which confirmed the successful expression of GST-AccMRJP1.This work provids a technical base for the utilization of MRJP1 with biological engineering.

The coding region of pre-pro-Acc-royalisin was amplified by PCR from cDNA library of the Chinese honeybee,Apis cerana cerana head,and was cloned into the vector pGEX-4T-2 for expression in Escherichia coli BL21.The expressed fusion protein,glutathione S-transferase(GST)-pre-pro-Acc-royalisin of 36 ku was obtained,which was cross-reacted with GST antibody accounting for up to 16.3% of bacterial protein.With the expressed products retrieved from the SDS-PAGE gels as antigen to immunize New Zealand white rabbits,the polyclonal antibody was prepared.With the purified recombinant GST-pre-pro-Acc-royalisin fusion protein as antigen,the high titers of the antibody was shown with ELISA analysis.The specificity of the antibody against the same antigens was then confirmed by Western blot.This study provides a new tool for the detection of antimicrobial of royal jelly,biological product quality of royalisin and resistance of honeybee.

Major royal jelly protein 1 (MRJP1) is the most abundant member of the major royal jelly protein (MRJP) family of honeybee. Mature MRJP1 cDNA of the Chinese honeybee (Apis cerana cerana MRJP1, or AccMRJP1) was expressed in Pichia pastoris. SDS-PAGE showed that recombinant AccMRJP1 was identical in molecular weight to the glycosylated AmMRJP1 from the Western honeybee (Apis mellifera). Western blots probed with anti-AccMRJP1 antibody demonstrated that recombinant AccMRJP1 and soluble protein of the Western honeybee RJ (AmSPRJ) contained immunoreactive MRJP1. The 57 kDa protein in AmSPRJ contained an N-terminal amino sequence of N-I-L-R-G-E, which is identical to that previously characterized in AmMRJP1. The molecular weight of recombinant AccMRJP1 was decreased from 57 to 48 kDa after deglycosylation, indicating that AccMRJP1 was glycosylated. The recombinant AccMRJP1 significantly stimulated Tn-5B-4 cell growth, similar to AmSPRJ and fetal bovine serum, and affected cell shape and adhesion to the substrate.