Major royal jelly proteins(MRJPs) were hydrolyzed with pepsin and trypsin,and the effects of substrate concentration,pH value,enzyme response time and proteinase concentration on the hydrolysis efficiency were analyzed,and the orthogonal test analysis of combined action of pepsin and trypsin were done.The enzymolysis products of MRJPs(H-MRJPs) were separated to prepare the angiotensin I-converting enzyme(ACE) inhibitory peptides by ultrafiltration technique.The results showed that the optimized technical parameters were as follows:enzymatic hydrolysis of MRJPs with 1% pepsin at pH 2.0 for 2 h,then 1% trypsin at pH 7.5 for 2 h at 37 ℃.The degree of hydrolysis and total nitrogen recovery of MRJPs under the optimal condition were 28.7% and 35.5%,respectively.No obvious protein bands were shown for enzymolysis products of MRJPs(H-MRJPs) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Three kinds of ACE inhibitory peptides,ranging from 1,1-5 and5 ku,were separated by ultrafiltration separation of H-MRJPs.The 50% inhibitory concentration(IC50) values of the three kinds of ACE inhibitory peptides were 0.33,0.61,and 1.09 mg·mL-1,respectively,which exhibited that the peptides with molecular mass 1 ku possessed most high ACE inhibitory activity.The results above provide a scientific basis for developing functional food of antihypertension.

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.

Bee venom phospholipase A2 (BvPLA2) 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 BvPLA2 (AccPLA2) 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 AccPLA2 fusion protein accumulated up to 5.32% of the total cellular proteins. The AccPLA2 fusion protein was cross reactive with the anti-AmPLA2 (BvPLA2 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 AmPLA2 in Western blot analysis. The PLA2 activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 μmol/(min·mg). In summary, the recombinant AccPLA2 protein, a native BvPLA2-like structure with corresponding biological activities, can be glycosylated in Tn cells. These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA2 in the pharmaceutical industry.

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.