Expression Of Cecropin AD In Newcastle Disease Virus Vector System And Its Bacteriostatic Effect

Human dependence on antibiotics leads to their abuse,which ultimately leads to the creation of large numbers of resistant bacteria and even super-resistant bacteria.Antimicrobial peptides have a unique mechanism of action that does not easily make bacteria resistant,so antimicrobial peptides are expected to be the best antibiotic alternative.Cecropin is the earliest discovered antibacterial peptide with good bactericidal/bacteriostatic effect,of which Cecropin AD（CAD）is one of them.Due to the high cost of artificial synthesis,there is an urgent need for suitable expression systems to express CAD antimicrobial peptide,so that they can be widely used in the treatment of bacterial diseases.In this study,we constructed and rescued a CAD-expressing Newcastle disease vector live virus vaccine candidate,which continuously produces CAD antibacterial polypeptides in the immune host to act on the site of infection and kill bacteria,thereby treating bacterial diseases.The main research contents and results of this project are as follows:1、Construction of full-length plasmids of recombinant Newcastle disease virus and virus rescueThe CAD and CAD₃gene fragments were inserted into the non-coding region between the P and M genes of the NDV TS09-C strain vector by In-Fusion homologous recombination,and the reverse genetic operating system was used to successfully rescue two recombinant Newcastle disease viruses r TS-CAD and r TS-CAD₃.The recombinant virus r TS-CAD can proliferate stably in SPF chicken embryos,but the recombinant virus r TS-CAD₃has CAD copy number loss when it is transmitted to the 5th generation and cannot be stably passaged.Through biological characterization analysis,the insertion of CAD and CAD₃genes between the P and M genes of NDV TS09-C did not change the weak virulence and proliferation characteristics of the parent strain.Indirect immunofluorescence assays confirmed that the recombinant Newcastle disease virus can successfully express CAD and CAD₃antimicrobial peptides in BHK-21 cells.2、Inhibition of bacteria by the antimicrobial peptide of cecropin AD expressed by Newcastle disease virusIn this study,three methods were used to verify the inhibitory effect of CAD antimicrobial peptides expressed by recombinant viruses on bacteria in vitro.The growth curve method was used to show that the recombinant virus group had the best bacteriostatic effect in the 2-4 h,which reduced the survival rate of S.aureus to47.0%-57.0%and 9.9%-18.8%,respectively.In the microplate inhibitory test,the inhibition rates of recombinant virus group against S.aureus were 79.6%-91.7%and79.7%-93.7%,respectively.The inhibition rates against E.coli were 87.5%-89.8%and 79.7%-87.5%,respectively.In the co-culture experiment with DF-1 cells,the inhibition rate of S.aureus by recombinant virus group could reach 74.5%-77.7%and30.0%-68.0%,respectively.The inhibition rates against E.coli bacteria were35.0%-45.0%and 8.0%-45.0%,respectively.In summary,the CAD and CAD₃expressed by the NDV vector system have a certain inhibitory effect on S.aureus（ATCC 29213）（G+）and E.coli（ATCC 25922）（G-）in vitro,and the inhibition effect on S.aureus is better.In addition,there was no significant difference in the in vitro inhibitory rate between r TS-CAD₃and r TS-CAD groups measured by different test methods.In order to prove the inhibitory activity of CAD antimicrobial peptides expressed by recombinant viruses in vivo,an animal model of mouse wound infection with S.aureus was established,and then two recombinant viruses,r TS-CAD and r TS-CAD₃,were injected subcutaneously into the wound skin.On the 7th day after inoculation,the number of S.aureus at the wound site was significantly reduced compared with the control group（P<0.01）,and the number of colonies in the r TS-CAD₃group was significantly less than that in the r TS-CAD group（P<0.01）;the size of the wound is significantly reduced;the pathological section of the skin at the wound showed local thickening of the epidermis and neutrophil infiltration of the skin tissue of the two treatment groups of r TS-CAD and r TS-CAD₃.With the passage of time,a large number of connective tissue hyperplasia and a large number of new capillaries were visible under the skin,and gradually entered the fourth stage of healing,while the skin structure of the control group was not clear,some skin necrosis,and slow healing.In summary,it is proved that the CAD antibacterial polypeptide expressed by recombinant virus can show obvious inhibitory effect on S.aureus in mice,and the inhibitory effect of r TS-CAD₃group is better than that of r TS-CAD group,and it also promotes the rapid healing of mouse wounds.3、Diversity analysis of chicken lung and intestinal floraIn this study,2-week-old SPF chicks vaccinated with r TS-CAD and r TS-CAD₃,7d-21d did not have significantly higher NDV antibody levels compared to NDV vector controls.After 16S sequencing of chicken lung flora,it was found that it was mainly composed of four major phylums:Bacteroidota,Firmicutes,Proteobacteria and Actinobacteriota.The analysis of sample community structure showed that both CAD and CAD₃expressed by recombinant virus could improve the richness of chicken lung flora.In addition,several significantly different microbiota,such as Enterobacteriaceae,Lactobacillaceae and Bifidobacteriaceae,were screened out by Kruskal Wallis algorithm analysis.The results showed that CAD and CAD₃expressed by recombinant viruses could significantly inhibit pathogenic bacteria such as Enterobacter and Bacteroides,and increase the microbiota abundance of probiotics such as Lactobacillus and Bifidobacteria.The recombinant virus expressing CAD₃inhibited pathogenic bacteria such as Enterobacter significantly higher than that in the r TS-CAD group（P<0.01）.After sequencing the contents of the chicken cecum,it was found that there were certain differences in the microbiota abundance of the intestinal flora at the phylum level in different experimental groups,and the microbiota abundance of the recombinant virus group was slightly higher than that of the TS09-C control group.Through data analysis,it was found that the abundance of anaerobic bacteria such as Bacteroides and Ruminococcaceae in the intestines of the two groups of recombinant virus was significantly lower than that of TS09-C control group（P<0.1）,and the r TS-CAD₃group was significantly lower than that of r TS-CAD group（P<0.01）,and beneficial bacteria such as Lactobacillaceae and Bifidobacteria were significantly higher than that of TS09-C control group（P<0.05）,and the r TS-CAD₃group was significantly higher than that of r TS-CAD group（P<0.01）.The above test results show that the recombinant virus can significantly inhibit pathogenic bacteria in the intestine of chickens,provide a favorable environment for the growth of probiotics,reduce the occurrence of intestinal diseases,and benefit intestinal health.In summary,this study constructed NDV recombinant virus successfully expressed CAD and CAD₃,the recombinant virus expressed cecropin AD antibacterial peptide for S.aureus（ATCC 29213）and E.coli（ATCC 25922）had obvious inhibitory effects,which helped mice to infect S.aureus.The healing of S.aureus wounds can colonize the respiratory tract and intestines of chickens and affect the number of flora in this area,inhibit the growth of some pathogenic bacteria,significantly increase the microbiota abundance of probiotics such as lactobacillaceae and bifidobacteria,and lay a foundation for exploring and expanding the application of NDV virus vectors and antibacterial peptides.