Recombinat Expression Of AccRoyalisin Gene From Apis Cerana Cerana And Its Antibacterial Activity

Royal jelly (RJ) is a glandular secretion produced by honeybees nurse to nourish honeybee larvae during their first three days of life and as the sole food for the queen during its life span. It contains rich biological active composition, and with much nutritional and physical functions. The Chinese honeybee (Apis cerana cerana) is a native honeybee in China, and its disease resistance is superior to the Western honeybee (Apis mellifera). Recent researches have shown that the disease resistance and the characteristics of non-perishable royal jelly are related to the peptide royalisin.Our laboratory has previously completed the expression of the Chinese honeybee peptide, AccRoyalisin and the preliminary studies on the antibacterial activity of its recombinant expression products in E. coli. This study focused on the enlarge fermentation tests of the prokaryotic expression, the large-scale expression of the products, and the separation and purification of the products. Based on these reserch, the systematic study of establishment of prokaryotic expression products on the inhibitory spectrum, minimum inhibitory concentration test (MIC) for representative strains, antibacterial stability experiments and antibacterial mechanism experiments were included.Firstly, adopting primers 5-P contained 6×His-EK DNA and 3-P, and AccRoyalisin-pGEM vector as the template, the recombinant AccRoyalisin was amplified with PCR reaction. Secondly, the PCR product was linked with GST whithin pGEX-4T-2 vector. Finally, using the recombinant vector, pGEX-4T-2-AccRoyalisin as template, the DNA fragment GST-6×His-EK-AccRoyalisin with 850 bp in length was amplified and inserted into yeast expression vector pPIC9K. The linilized AccRoyalisin-pPIC9K was transformef into of Pichia yeast GS115 with electroporation. Through PCR identification with the DNA genome extrcted from the yeast clones, the recombinant yeast with purpose gene was identified. The recombinant yeast clones were induced to express peptides. SDS-PAGE analysis showed that the expression product possessed a specific band wih 30 kDa in molecular weight. Western blot analysis using the GST polyclonal antibody as the first antibody proved that GST-AccRoyalisin was expressed in yeast successfuly. A a single peptide band was purified from the expressed product.Antibacterial detection showed that the recombinant AccRoyalisin expressed in yeast possessed inhibitory activity against Gram-positive bacterias, such as Bacillus subtilis and Micrococcus lysodeikticus.