Tachyplesin Ⅰ-human Lysozyme Expression In Pichia Pastoris, Condition Optimization For Large-scale Fermentation And Analysis Of Biological Activity

Antibiotic resistance has become a global issue. The abuse of antibioticspromoted the emergence of antibiotic-resistant bacteria. The emergence of “superbacteria” announced that the human has entered the “post-antibiotic era”. Therefore,the development of new antimicrobial agents, which is used to replace of antibiotics,has very important significance. Hunam Lysozyme (HLYZ) catalyzes hydrolysis tothe β (1-4)-linked N-acetyl muramic acid and N-acetyl glucosamine of peptidoglycanin bacterial cell wall with strong specificity, but the high dose for using restricted itsdevelopment. Through the artificial modification we can enhance the antibacterialactivity of human lysozyme, which will improve its sterilization efficiency.Anti-microbial peptides is the small molecule polypeptide, which is the best candidatemolecule associated with human lysozyme.In this study, we selected the flexible and rigid linker as the linker peptides, thendesigned and synthesized the Tachyplesin Ⅰ-human lysozyme fusion gene. Afterthat we constructed a eukaryotic expression vector, transformated into Pichia pastoris.After induced by methanol, we analyzed the antibacterial activity of the supernatantand determined that the antibacterial activity of the fusion protein linked by A(EAAAK)2A is stronger than the other one.We selected the pH of culture medium, methanol dosage and inducing time thesethree factors at three levels to design orthogonal experiment with the model of L9(34)for the optimization of expression condition. Fermentation supernatant was used toanalyze the antibacterial activity for studying the various factors on the expressionlevels of the fusion protein. Ultimately, we determined the best fermentation conditionas follows:72hours for expression,2%methanol and fermentation liquid with the pH5. Then we did a small-scale experiment in the7L fermenter and used membranefiltration, ultra-filtration tube thickening and affinity chromatography method forpurifying the fusion proteins and the purity was up to92.1%. The analysis ofantibacterial activity with fermentation supernatant against Staphylococcus aureusdemonstrated that the bacteriostatic ring diameter was28.5mm. Finally, with the comparison by testing the MIC and agar diffusion test of thefusion protein and huma lysozyme against the selected10kinds of strains, we foundthat the antibacterial activity of the fusion protein was stronger than that of the humanlysozyme, with a minimum1value12.5μg/mL. In order to research antibacterialmechanism of the fusion protein, we chose the Staphylococcus aureus andEscherichia coli as the experimental strains and used scanning electron microscopy toanalysis, which showed that the fusion protein synthesized the antibacterialmechanism of Tachyplesin Ⅰand human lysozyme. In addition, in vivo experiments,we selected mice as the experimental animal to establish a septicemia model withStaphylococcus aureus, and took the protecting rate and colony forming units of theblood as the standards to determine the best time and dosage, which were30minutesafter attacking and50μg, respectively. And in vivo treatment trial showed that theprotection rate of fusion protein was up to100%.