Recombinant Tachyplesin I: Large Scale Preparation, Biological Effects And Mechanism In Vitro And In Vivo

On April 7, 2011, World Health Day, World Health Organization called for"Combat Drug Resistance: No Action Today, No Cure Tomorrow". The increasing antibiotic resistance problems were largely due to irrational use even abused of antimicrobial agents as feed additives. Therefore, the drug research of new, safe, no drug residue, no antibiotic resistance is an important and urgent scientific problem. Since the discovery of antimicrobial peptides (AMPs) in 1972, it has been a potential solution to the dearth of novel antibiotic development. AMPs have shown activity against a wide variety of pathogens with new mechanisms. Recent studies had also shown that antimicrobial peptides could not only directly kill pathogenic bacteria invade bodies, but also display a variety of beneficial effects on the different stages of the host innate immune response. Based on the current trends in domestic and international research, AMPs was expected to become a substitute for antibiotics in animal feeds. The research and application of AMPs to the Livestock and poultry breeding industry will be an important measure to eliminate antibiotic residues, which will finish the embarrassing situation of adding antibiotic to the feed additives over the years, and create a new concept of green farming. However, the research on the mechanism of action of AMPs and the interaction of the AMPs with the animal body are the key bottleneck on large-scale application of the AMPs as anti-microbial agents and feed additives, are the theoretical basis for market approval of the AMPs, and are the important guarantee for improving the public awareness and confidence to the AMPs.The constitutive expression of Tachyplesin I (TP I) with glucose as inducer in Pichia pastoris has been successfully established in this study. The response surface methodology was applied to optimize the fermentation conditions. After optimization, the fermentation was carried out in a 7.5L bioreactor. Using high-performance liquid chromatography to detect and quantitate TP I, the result showed that the secreted TP I reached levels of 40 mg/L in fermentation supernatant. The ESI mass spectrometry results showed the molecular weight of the fermentation product was 2263.79 d, consistent with the theoretical calculation.The recombinant TP I displayed broad-spectrum antibacterial activity against 14 genus 61 strains bacterium using the agar well diffusion method and susceptibility test, which showed strong inhibitory to Gram-negative E. coli, Salmonella, Campylobacter jejuni, Proteus and Gram-positive Staphylococcus aureus, Bacillus subtilis, Streptococcus and Enterococcus feces (Inhibition zone diameter>20 mm). The antibacterial effect of TP I to gram-positive was predominance, the minimum inhibitory concentration (MIC) of which to Staphylococcus aureus and Bacillus subtilis was 0.63μg/mL. And the TP I had the same inhibitory ability to fluoroquinolone resistant and sensitive Staphylococcus aureus strains, which showed that the sensitivity of bacteria to TP I was not affected by the sensitivity to antibiotics. TP I also achieved the same therapeutic effect as antibiotics on Staphylococcus aureus epidermidis infection in mice model.To develop monoclonal antibodies against TP I, the synthesized TP I was chemically conjugated with the carrier protein BSA and then injected into BALB/c mice. Five MAb, effectively in detecting the native TPI, named 2D8, 3B8, 5H2, 6B12 and 8F5 were obtained. Isotyping of all obtained MAbs indicated that the MAbs of 2D8, 3B8, 5H2 and 8F5 belong to IgG1, the 6B12 belong to IgG2a respectively. Specificity assay showed 8F5 had almost the same level of specificity to Natural TP I, recombinant TP I, synthesized TP I and TP II respectively, but had not cross-react with control peptides. These results suggested that the synthetic AMPs conjugate can elicit antibodies against native AMPs and the MAb we got can be used to detect antimicrobial peptide.We also measured the antiviral activity of TP I and characterized its antiviral mechanisms to Newcastle disease virus (NDV). TP I was active against NDV infections in vitro. Using 10μg/mL TP I in 0.1 MOI NDV/cell, the plaque reduced 65% and the virus load reduced 57% measured by fluorescent quantitative RT-PCR. Vero cells were labeled fluorescent probe DiBCA and the changes of membrane potential measured by flow cytometry. The results demonstrated that cell membrane infected by NDV was in the state of hyperpolarization, and TP I could significantly intervene this hyperpolarization. Vero cells membrane labeled NBD-C6-HPC and confocal microscope were used to study the changes of Vero membrane fluidity which infected by NDV and TP I on the virus infection. The results showed that the membrane fluidity decreased significantly post NDV, and TP I could improve the membrane fluidity when viruses attachment. Above results showed protective effect of TP I on target cells against virus infection.TP I freeze-dried powder of expression supernatant was feed as additive to study the effect on the growth performance in chickens. After administered 200 g/t TP I powder, the average daily weight gain, the index of thymus and fabricius of the experimental group chickens was significantly higher than those of the control group chickens, and had no significant difference with antibiotic group. When compared to the control group, the intestinal villi arranged more closely and the villi branch was increase. Immunohistochemistry results showed that TP I was located in epithelium mucosae, crypts of lieberkuhn and intestinal villi especially. The results demonstrated that TP I could enhance the growth performance and improve immune function of chickens.Comparative metagenomics of intestinal contents was established by denaturing high performance liquid chromatography (DHPLC) which could distinguish the intestinal flora 16s rRNA gene fragment. The DHPLC results showed the species in middle dose group intestinal tract was simpler than the control group, and the species of the blank group was multiplicity. Accordingly, the TP I could inhibit the growth of some types of bacteria, and improve the growth of other bacteria. In this study, we have successfully established constitutive expression of Tachyplesin I in Pichia pastoris. After optimization, the secreted TP I reached levels of 40 mg/L in fermentation supernatant. The recombinant TP I displayed broad-spectrum antibacterial activity against 14 genus 61 strains bacterium especially to gram-positive. Then we evaluated the therapeutic effect of the TP I on Staphylococcus aureus epidermidis infection in mice model. We also measured the antiviral activity of TP I to NDV and characterized its antiviral mechanisms. The TP I could significantly protect target membrane hyperpolarization of cells infected by NDV and improve the membrane fluidity. The appropriate dose of TP I just like antibiotic could enhance the growth performance and improve immune function of chickens. The TP I could impact on bacteria species in the intestinal tract. Accordingly, the TP I could inhibit the growth of some types of bacteria, and improve the growth of other bacteria. Our study provided an experimental and theoretical basis for the applications of TP I as anti-microbial agents and feed additives.