The objective of this study was to construct an improved thioredoxin fusion protein expression system, and express the cathelicidin-derived peptide, Lf-CATH2. The improved fusion vector Lf-CATH2-pET32α(-TS) was successfully constructed by firstly deleting the thrombin site and S tag from the pET-32α vector, then inserting the Lf-CATH2 plus a thrombin site instead. Afterwards, Lf-CATH2 was expressed in Escherichia coli as fusion protein. After the cleavage by thrombin, Lf-CATH2 was released and subsequently separated using affinity chromatography. The antimicrobial activity of purified Lf-CATH2 was also examined. The improved expression vector significantly increased enzyme cleavage efficiency by 37%, and Lf-CATH2 could be expressed in high yield and maintain the biological activity. This novel thioredoxin fusion protein expression system enables a quick production of high-yield bioactive cationic peptides like cathelicidins.
Cathelicidins ; biosynthesis ; Chromatography, Affinity ; Escherichia coli ; Genetic Vectors ; Recombinant Fusion Proteins ; biosynthesis ; Thioredoxins ; genetics

Objective:To study the frequency of electrical stimulation affect the atrophy recovery of mouse soleus muscle.Method:Sixty mice,30 of them were randomly divided into blank control group (Con) and atrophy model group (Atro) and validated this model after 1 week.The other 30 mice were randomly divided into three groups:the natural recovery group (Rec),the exercise intervention group (Exer),the electroacupuncture intervention group (Elec) after making them atrophy.Atrophy model group were plaster fixed one week.Natural recovery group was natural fed two weeks after removing the plaster.Exercise intervention group runs 1 hour at 16m/min speed two weeks.EA intervention group were stimulated 15min daily for 2 weeks on hind limb,with pulse duration 0.35ms,frequency 50Hz,for 2 weeks.Frozen sections and Immunofluorescence were used to measure the cross-sectional area of muscle fibers.ATPase staining was used to detect the muscle fiber type changing,and Western Blot to detect soleus muscle Troponin I-SS (TNNI1) and Troponin I-FS (TNNI2) protein expression.Result:The soleus muscle in Atro group showed significant change in cross-sectional area and wet weight compared with Con group(P＜0.01),The expression of TNNI1 and TNNI2 protein was also significantly different from that of Con group (P＜0.05).Compared with the Rec group,the cross-sectional area of slow muscle and fast muscle in the Exer and Elec groups significantly increased (P＜0.05),TNNI1 protein expression significant ly increased (P＜0.01),TNNI2 protein was no significant difference detected(P＞0.05).Conclusion:The middle low frequency electroacupuncture intervention can effectively increase the cross-sectional area of muscle fibers,in particular,increase the TNNI1 and TNNI2 protein expression of slow muscle fibers.It is as effective as exercise intervention to promote recovery of disuse muscular atrophy.

Based on the cathelicidin family antimicrobial peptide Hc-CATH derived from sea snake, the Hc-16 and Hc-15 of 16 and 15 amino acid residues, were designed. By using CCK8, minimal inhibitory concentration, ELISA and bio-layer interferometry assays, their cytotoxicity, antibacterial activity, anti-inflammatory activity, and LPS neutralization activity was examined. Compared with Hc-15, Hc-16 had lower cytotoxicity and better broad-spectrum antibacterial activity against pathogens including clinically resistant bacteria, with the minimum inhibitory concentration of only 4.69 μg/mL. Hc-16 inhibited the expression of inflammatory cytokines of TNF-α and IL-6 induced by LPS, so as to significantly reduce the inflammatory response induced by infection. In addition, structure-activity relationship studies have shown that the phenylalanine at the C- and N-terminals of Hc-16 played a crucial role in its antibacterial and anti-inflammatory activity. Altogether, the designed Hc-16 has an excellent prospect to be developed into a novel antibiotic.
Animals ; Anti-Bacterial Agents/pharmacology* ; Anti-Infective Agents ; Hydrophiidae ; Microbial Sensitivity Tests ; Pore Forming Cytotoxic Proteins