The Study Of The Antibacterial Activity Of Recombinant Human Lysozyme And It’s Effect On Bacterial Community And Diversity In The Mice Intestinal Tract

Bovine mastitis is an important disease affecting the development of dairy productionindustry. As the lysozyme has significant antibacterial properties to almost all thegram-positive bacterium and some gram-negative bacteria, it is an effective new way tocultivate human lysozyme gene transgenic cows to prevent bovine mastitis. Although humanlysozyme gene transgenic cows has some certain ability to resist bovine mastitis, thelysozyme which efficiently expressed in the breast will whether affect the digestive tract florabacteria community structures of the calves and even human through feeding the milkremains unclear, and the research reports about this are few. In this study, we tested theantibacterial activity of recombinant human lysozyme (rhLYZ) in vitro through the flatdiffusion method and gradient dilution method, and detected the mice intestinal bacteriastructure diversity and dynamic changes after fed with certain dose of rhLYZ throughdenaturing gradient gel electrophoresis (DGGE) technology.1. Using the flat diffusion method, we test rhLYZ antibacterial activity with severalpathogenic bacteria strains and probiotic bacteria strains in vitro, the comparison control ofcommercialization of gallinaceous egg white lysozyme is added. Simultaneously we detectthe MIC and MBC of recombinant human with lysozyme each bacteria using gradient dilutionmethod. Recombinant human lysozyme and commercialization of gallinaceous egg whitelysozyme are added onto MH diffusions which contain certain volume of bacteria after stepdiluted respectively. Through measuring the bacteriostatic circle diameter, we can determinetheir antibacterial activities preliminary. The results showed that recombinant humanlysozyme have notable higher antibacterial activity than the commercialization ofgallinaceous egg white lysozyme, low mass concentrations of recombinant human lysozymecan inhibit the growth of pathogenic bacteria, especially more sensitive with StaphylococcusAureus and Escherichia coli, the diameter of antibacterial circle is up to15mm~18mm and amost high score is27.5mm with Pasteurella multocida. This implies that rhLYZ has a strong inhibitor bacteria activity with pathogenic bacteria like Staphylococcus Aureus, Escherichiacoli, Pasteurella multocida, et al.2. The bacterial community and diversity in the mice intestinal tract were studied byusing16s rDNA based DGGE and some common and special bands were identified, in orderto investigate the effect on the mice intestinal flora structure by feeding with differentconcentration dose of rhLYZ. The results showed that the microbes of all the mice feces hadan increasing trend as the mice growed. After feeding the mice with rhLYZ, the microbialdiversity of1.0mg/mL dose group significantly reduced (P<0.01) while0.5mg/mL dose group,0.25mg/mL dose group and0.1mg/mL dose group had no significant difference (P>0.05)compared with the control group; UPGAMA clustering analysis results showed that there wasa lowest similarity of the intestinal flora structure (31.9%) bettween1.0mg/mL dose groupand the control group, the similarity of0.5mg/mL dose group,0.25mg/mL dose group and0.1mg/mL dose group had a nearly similarity of40%compared with the control group, whichindicated that feeding the mice with rhLYZ have some certain effects on animal intestinalbacterial community and diversity, and these effects appeared dose-related. In addition, thepredominant bacterial in each dose group of mice intestinal tract remain unchanged, whichmainly contained Bacteroides sp., Staphylococcus equorum and Staphylococcus lentus,Lactobacillus johnsonii strain and Lactobacillus reuteri strain.