Sudies On The Expression Of Acc-royalisin From Royal Jelly Of Chinese Honeybee In Escherichia Coli And Its Antibacterial Activity

The Chinese honeybee (Apis cerana cerana) is native honeybee in China and anti-disease effectively than East honeybee (Apis mellifera). Roya Jelly is producted by nurse honeybee, milkiness and has lots of biological activity. RJ are nutritional but metamorphose hardly, it is depend on the antibacterial component. MAR is an antibacterial peptide, belong to bee definsin species, as reported it have anti-G+bacteria effect. This reacher finished pre-pro-Acc-royalisn and mature Acc-royalisin expression in E. coli BL21 and antibacterial analysis.1. Expression of royalisin from royal jelly of Apis cerana cerana in Escherichia coli and preparation of its polyclonal antibodyThe part of PPAR was amplified from Apis cerana cerana cDNA library and subcloned into the expression vector pGEX-4T-2 with a glutathione S-transferase (GST) at the N-terminus. Fusion protein GST-PPAR was expressed in E. coli BL21. SDS-PAGE showed that the expression product was 46 ku which was accordance with its expected size. Layer scanning showed that the amount of GST-PPAR accumulated up to about 16.3%of the total bacterial proteins. Western blotting with GST Monoantibody confirmed the correct expression. Also purified GST-PPAR was injected subcutaneously to immunize New Zealand white rabbits to prepare polyclonal antibody specific for PPAR. The titer of the antibody was evaluated by ELISA analysis and its specificity was confirmed by Western blot with purified GST-PPAR which means the raised polyclonal antibody could be used to detect PPAR.2. Expression of pro-pre-royalisin and purification analysis of expressed proteinThe gene clone, expression and analysis of the soluble proteins are accomplished in this research. Western blotting with GST Monoantibody confirmed the correct expression. Expressed fusion protein, glutathione S-transferase GST-PPAR of 31.6 kDa is obtained, which accounting for up to 18.6%bacterial protein. In addition, nearly 100%of GST-MAR was soluble protein. 3. The purification of GST-pro-pre-royalisin and GST-royalisinBoth GST-PPAR and GST-MAR protein are purified by GenScript Corporation kit. We gain 0.0134g and 0.0206g protein from 500 mL bacterial culture respectively. Western blotting with GST Monoantibody confirmed the correct expression. Incised by Thrombin, GST-MAR released MAR.4. Antibacterial analysisBoth 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-PPAR and GST-MAR fusion protein.