To improve the expression level of tmAMP1m gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMP1m by conditions, such as culture temperature, inducing time and the final concentration of inductor Isopropyl beta-D-thiogalactopyranoside (IPTG). We analyzed the optimum expression conditions by Tricine-SDS-PAGE electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. The results suggest that when inducing the recombinant plasmid with a final IPTG concentration of 0.1 mmol/L at 37 degrees C for 4 h, there was the highest expression level of fusion protein HIS-TmAMP1m in Escherichia coli. Under these conditions, the expression of fusion protein accounted for 40% of the total cell lysate with the best antibacterial activity. We purified the fusion protein HIS-TmAMPlm with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices. Western blotting analysis indicates that the His monoclonal antibody could be specifically bound to fusion protein HIS-TmAMPlm. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET30a-tmAMP1m. The fusion protein HIS-TmAMP1m had better stability and remained higher antibacterial activities when incubated at 100 degrees C for 10 h, repeated freeze thawing at -20 degrees C, dissolved in strong acid and alkali, or treated by organic solvents and protease. Moreover, the minimum inhibitory concentration results demonstrated that the fusion protein HIS-TmAMP1m has a good antibacterial activity against Staphylococcus aureus, Staphylococcus sp., Corynebacterium glutamicum, Bacillus thuringiensis, Corynebacterium sp. This study laid the foundation to promote the application of insect antimicrobial peptides and further research.
Animals ; Anti-Infective Agents ; pharmacology ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Insect Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Tenebrio ; chemistry

Background:Early administration of enteral nutrition can improve intestinal mucosal barrier in patients with severe acute pancreatitis (SAP),reduce infection and complications;but when should the enteral nutrition be started is still controversial. Aims:To study the effect of different initiation time of enteral nutrition on elderly obese patients with SAP. Methods:The clinical efficacy,hospitalization time,hospitalization cost and the risk of complication were analyzed retrospectively in the two groups of patients with either ultra-early enteral nutrition (group A)or early enteral nutrition (group B),as well as the serum levels of inflammatory cytokines before and after treatment. Results:No significant difference in total effective rate was found between group A and group B (90. 9% vs. 79. 2%,χ2 = 1. 227,P > 0. 05). Compared with group B,the recovery time of blood and urine amylase,and hospitalization time in group A were significantly shortened and the cost of hospitalization was significantly decreased (P < 0. 05). The time of abdominal pain, bloating,vomiting and fever in group A was significantly shorter than that in group B (P < 0. 05). After 14 days of treatment,levels of IL-6,TNF-α and CRP in group A were significantly lower than those in group B (P < 0. 05). The incidence of infection was significantly lower in group A than in group B (P < 0. 05). Conclusions:Compared with early enteral nutrition group,ultra-early enteral nutrition for elderly obese patients with SAP has better efficacy,and has a shorter hospital stay and lower hospitalization cost.