Optimized Expression And Antibacterial Activity Analysis Of Antimicrobial Peptide CecropinXJ Of Bombyx Mori

Antimicrobial peptides is a class of defensive peptide active substances, an importantcomponent of innate immunity, produced by immune defense system for against exogenouspathogens. Antimicrobial peptides are usually composed by12to100amino acid residues,containing many hydrophobic amino acids, and most of them are amphiphilic cationic peptides.According to the composition and structural characteristics of amino acids, antimicrobial peptidescan be divided into cecropins, defensins, magainins and melittins. Antimicrobial peptides is aenvironmentally friendly biologic agent, non-toxic side effects, no residue and no bacterialresistance, with many advantages, such as thermal stability, good water solubility, broad-spectrumbactericidal even for fungi or protozoa. Hence, the application of antimicrobial peptidesgradually become a research focus area, and has potential commercial value in productsapplication and development areas.CecropinXJ gene cloned from Aksu Xin-silkworm NO.3, it has been successfully expressedin prokaryotic and eukaryotic systems. CecropinXJ belong cecropin-B class family, has anamphiphilic α-helix region,26amino acid signal peptide, mature peptide37amino acids,intense heat or acid stability, broad spectrum antibiotic, and even has cytotoxicity to a variety ofcancer cells. So, it has potential applications in the development of the food industry anddisinfection products.The study was supported by optimizing conditions for expressing CecropinXJ, study ofimpact indicators and signs of organs and tissues of mice using mice fed methods, preliminarydetect CecropinXJ toxicological effects, and further the potential commercial value of CecropinXJantibacterial product were evaluated.The study includes three parts:1. Optimized Expression and Antibacterial Activity Analysis of Antimicrobial PeptideCecropinXJ of Bombyx moriBy optimizing different medium C:N ratio, different induction times and differentconcentrations of inducer, the final results showed that the optimum conditions for prokaryoticexpression is optimized temperature37℃, medium N:C1:4, induction time4h at inducerconcentration1.28mmol/L. After sonication, the supernatant was taking protein fusion protein expression accounted for over50%of the total bacterial proteins, fusion protein bandscorresponding purified up to80%by affinity purified. CecropinXJ fusion protein amounted to10mg/L with optimal conditions mentioned. Optimal conditions for prokaryotic expression andpurification: The concentration of binding buffer was50mmol/L, the concentration of the washbuffer heteroaryl100mmol/L, the concentration of the elution buffer was250mmol/L.CecropinXJ can effectively inhibit bacteria and fungal growth by CecropinXJ bacteriostasis.2. Effect of Antimicrobial Peptide CecropinXJ from Bombyx mori on Physical Signs Indexesand Organs in MiceStudies shown that high purity large doses (40mg/kg/d, purity85%) CecropinXJ has noinfluence on the growth, weight, and other organ indexes of mice by detecting sign indicators andhistopathology of stomach and liver tissue fed with CecropinXJ indicators and signs of mice fedstomach and liver tissue, but for liver indexes. no significant effect on organ index was observedwith other dosage CecropinXJ in mice; Paraffin sections showed the stomach and liver of micehad no significant toxicological and pathological changes response to unpurified total protein, buthigh-purity high-dose (40mg/kg/d, purity85%) of CecropinXJ effected mouse liver toxicologicaland pathological changes.3. Evaluation of Commercial Application and Development of CecropinXJThe study of CecropinXJ influences on preservative effect of freshly squeezed juice showedthat CecropinXJ can effectively inhibit the growth of bacteria, keep fresh juices original nutrientsand flavor, extend shelf life With an appropriate concentration of CecropinXJ adding to freshlysqueezed juice. CecropinXJ has a very obvious inhibitory effect in both of tomato juice and grapejuice, and presents a different dynamic variations antibacterial form, maybe interaction betweenthe food matrix with relevant. its biological activity could effected by many factors, such ascomponennts, pH and microflora, and so on, but the specific reasons for further study anddiscussion.Based on CecropinXJ’s broad-spectrum antimicrobial activity, we conducted a dailyapplication preliminary exploration with bacteria on the surface of palm. Inhibitory concentrationand plate count showed that the gradully strong suppressing bacterial capability with CecropinXJconcentration gradully increase, there was a significant difference compared to the control group(p<0.05). Property identification results of bacteria genus inhibited by CecropinXJ show that CecropinXJ can significantly inhibit the growth of the palm surface of gram-negative bacteria.This study provides a theoretical basis that the CecropinXJ could use for development andapplication of life and medical sterilization product.