Construction Of Recombinant Lactobacillus Casei Constitutively Expressing Porcine Antimicrobial Peptides PF1-PR39 And Analysis Of Biological Activities

Antimicrobial peptides widely exist in the biosphere, in addition to having a broad-spectrum antimicrobial activity, they also have anti-bacterial, anti-viral, anti-fungal, anti-parasitic anti-protozoa and anti-cancer cell functions, thus playing an important role in the natural immune system of the organism. Antimicrobial peptide PR39 and PF1 is proline-rich antimicrobial peptides of cathelicidin family. Lactobacillus are probiotics which can colonize in the intestinal tract, they have much more advantages in expressing foreign proteins than other prokaryotic or eukaryotic. Furthermore, this constitutive expression system of recombinant bacteria do not need additional induce agent, which makes it more suitable for mass production and has good practical value and development prospects.In this study, based on the gene sequence of PF1 and P R39 published on Gen Bank, we designed our recombinant gene sequence. We used a rigid linker to connect PF1 and PR39, added a myc protein tag in the preceding sequence for detecting the protein expressio n, optimized the sequence according to Lactobacillus codon bias tropism and then synthesized PF1-PR39. PF1-PR39 with restriction sites of Xho I and Bam H I was amplified with primers P1, P2 and synthetic gene as a template, and was connected with p MD19-T Simple vector. After digestion with Bam H I and Xho I, the PF1-PR39 gene fragment was recycled and cloned into PPH-p PH612 constitutive expression vector. Positive recombinant plasmid was was transferred into the Lactobacillus casei ATCC 393(L.casei 393) to obtain the recombinant Lactobacillus casei which could express antibacterial peptide PF1-PR39 constitutively.To investigate the recombinant protein PF1-PR39 whether could improve animal growth performance and to explore the potential in vivo antibacterial a ctivity and protective effects, totally 45 female BALB/c mice of SPF with the weight of 14-17 g were assigned into 3 groups with 15 in each group. Experimental group were with 200 μL 1 × 109 CFU recombinant Lactobacillus casei PPH-p PG612-PF1-PR39 / L393 every day, 200 μL 1 × 109 CFU PPH-p PG612 / L393 for vector group and 200μL MRS for media group. At the beginning of the experiment, and on the 14 th and the 21 th day, we detected the weight of mice to calculate the average daily feed intake(ADFI), the average daily gain(ADG) and feed/gain(F / G). For each group of mice, blood samples were collected on the 7th and 14 th day, and on the 21 th day, 3 mice from each group were sacrificed, blood samples were collected and intestinal mucus was scraped from the small intestine. Total RNA was extracted with small intestine tissue, real-time PCR method was used to detect the expression level of the corresponding cytokine. The content of IgG and Ig M in blood samples and s Ig A in intestinal mucus was detected by ELISA. In each group, the remaining mice were divided into two groups and infected orally with ETEC K88+ and Salmonella typhimurium, respectively, and then we observed whether recombinant strain have protective effect on infected mice. The experimental mice had more lymphoid blood leukocytes, lymphocytes and higher lymphocyte ratio than the MRS group without significant difference. The concentration of Ig G and Ig M of the experimental group mice was higher than the other mice, in which the Ig M changed significantly(P <0.05), while s Ig A in intestinal mucus slight increase without significant change. RT-PCR results showed that the TNF-α and TGF-β expression levels of experimental group of mice have significantly improved, while expression levels of IFN-γ, IL-4, IL-6 decreased without significant difference. Recombinant bacteria expressed PF1-PR39 could protect mice infected with enterotoxigenic Escherichia coli and Salmonella to a certain degree. The protective effect on mice was up to 83.3% and 66.7%, respectively.In summary, recombinant Lactobacillus casei expressing PF1-PR39 has antibacterial activity in vitro. Supplementing mice with the recombinant strain can mitigate the pathogenic effect of pathogenic bacteria on mice and improve their growth performance as well as the immunity of mice. This study provide feasibility basis for genetic engineering production of antimicrobial peptides as probiotics.