Expression Of Plectasin In Pichia Pastoris And Its Effects On Rats Intestinal Health And Immune Function

Utilization of various antibiotics in livestock industry promote the growth of animals and decrease its susceptibility to diverse infectious diseases. However, their continuous use have led to the emergence of drug resistance and increased the risk of residues in livestock product. Currently, the research has been focused on developing of substitutes for antibiotic. Compared with conventional antibiotics, antimicrobial peptides with no drug resistance and satisfactory inhibitory effect is receiving attention gradually. Plectasin, which is produced by the fungus Pseudoplectania nigrella, is the first fungus-derived defensin. Studies showed that plectasin with strong activity against Gram-positive bacteria. However, production of plectasin from natural microorganisms is still not commercially feasible because of separation process complex and high-cost. To increase its production, a multimer form of plectasin from Pseudoplectania nigrella was expressed in the Pichia pastoris X-33 strain. In addition, the antimicrobial activity, pH stability and proteolytic resistance of recombinant plectasin were fully characterized.Experiment 1:Expression of the multimer form of Ple gene in P. pastorisA multimer form of Ple gene from Pseudoplectania nigrella was previously cloned in our lab, and was successfully expressed in E. coli. In this study, the multimer form of Ple gene (1,002 bp) was inserted into pPICZaA vector and subsequently expressed in P. pastoris. The selected P. pastoris strain was named PPle and yielded Pie 143 μg/mL under the control of methanol inducible alcohol oxidase 1 (AOX1) promoter. The recombinant Pie estimated by SDS-PAGE to be 41 kDa.Experiment 2:In vitro antimicrobial activity of the purified recombinant PieThe recombinant Pie secreted by PPle and purification by Ni2+-NTA affinity chromatography. In vitro, studies showed that the purified Pie efficiently inhibited the growth of several gram-positive bacteria such as the Streptococcus suis and Staphylococcus aureus. The S. suis was the most sensitive bacteria to Pie, with a minimum inhibitory concentration (MIC) of 4 μg/mL. Importantly, the recombinant Pie exhibited resistance to pepsin and maintained antimicrobial activity over a wide pH range (pH 2.0 to 10.0). The results showed that the recombinant Pie developed by our lab has the strong stability, which suggested that the recombinant Pie could be one alternative to antibiotics in animal production.Experiment 3:Effects of recombinant plectasin on intestinal health and immune function in ratThe experiment was conducted to investigate the effects of the Ple on immune function and intestinal health, and evaluate its application prospect in animal production. 60 healthy SD rats were randomly divided into 6 groups (n=10). The rats were feed individually and treated by intraperitoneal injection of Ple, vancomycin and the culture supernatant (control), consecutively. Half of the rats were challenged by S.aureus (other half as control was injected with sterile saline). The rats were reared for another two weeks until slaughtered for sampling. Results showed that S.aureus challenge had no significant effect on the immune organ index, but reduced the levels of IgM and IgA (0.05<P<0.10), injection of plectasin and vancomycin can increase the serum immunoglobulin levels and improve the immune function of rats; Moreover, S.aureus challenge significantly decreased the duodenum villus height and sIgA levels, and increased the amount of S.aureus in cecum (P<0.05). However, plectasin administration can alleviate the negative impacts of S.aureus, and its beneficial effect is better than vancomycin.In summary, the multimer form of Ple gene was successfully expressed in P. pastoris; The expression product (recombinant Ple) has strong inhibitory action to the gram-positive bacteria, especially S. suis and S. aureus; Injection of Ple can improve the immune function and intestinal health in rats, and the recombinant Ple has the potential to replace the vancomycin in the treatment of S. aureus; All these features described that the recombinant Ple could be served as an ideal substitutes of antibiotics used in animal production.