Polymorphism Analysis Of Sheep Antimicrobial Peptide GNLY Gene And The Effect Of Synthetic Peptide On Mastitis Model In Mice

Antimicrobial peptide is a kind of polypeptide widely existing in organisms.Its molecular weight is small and its chemical properties are stable.Antimicrobial peptides have high level of anti-bacterial,anti-tumor,anti-fungal,anti-parasitic,and anti-viral effects.Their unique mechanism of action makes bacteria resistant to drug resistance and has become a hot topic for scientists.Granulysin(GNLY),like most antimicrobial peptides,kills a variety of microorganisms and parasites and participates in the immune response.It is an important component of the body's natural immune system.There are few studies on sheep antibacterial peptide GNLY,and it is unclear whether the gene is different in sheep of different breeds.Purpose: In this study,we sequenced the GNLY gene CDS regions of Suffolk sheep,Merino sheep and Hu sheep in Xinjiang,to analyze the SNP polymorphisms of the GNLY gene in different breeds,and used bioinformatics methods to predict GNLY secondary levels.Inferring the relationship between amino acid sequence changes,protein structure and function;synthesized several GNLY polypeptides,measured the biological activity of synthetic peptides in vitro and other biological activities;selected peptides with strong activity in vitro to carry out the mastitis model in mice treatment,determinated the role of synthetic peptides in vivo,provided a scientific basis for the development of animal anti-infective drugs.Methods:(1)?RNA was extracted from peripheral blood of three breeds of sheep and reverse transcribed into cDNA followed by PCR amplification.PCR products were sequenced.The amino acid sequence of GNLY was analyzed by software to predict the secondary structure and the polypeptide was designed based on amino acid sequence of the sheep GNLY and selected polypeptides of different lengths,and designed retain one ?-helix and LOOP structural at least.(2)?The antibacterial activities of synthetic peptides on Escherichia coli,Salmonella,Staphylococcus,Staphylococcus aureus were determined and its inhibition growth to human esophageal cancer cells(EC109)and human renal carcinoma cells(X786-0)were detected as well.(3)?The toxicity of synthetic peptides was verified by rabbit red blood hemolysis test;After parturited 8 to 10 days of KunMing mice,were randomLy divided into 4groups and recorded as PBS treatment group,peptide treatment group,ampicllin treatment group,and blank control group.Except for the blank control group,all the other mice were injected with Staphylococcus aureus,and treated with PBS,peptide,and ampicllin respectively after 3 hours.After 24 hours,all the mice were killed and some tissues were taken for pathological section observation.Another tissues were subjected to detected CFU count and TNF-?.Results:(1)?The experiment showed that the secondary structure of sheep GNLY contains 8?-helices and 6 LOOP structures;according to the sequencing results of PCR products,the nucleotide sequence of GNLY gene in three breeds of sheep have slightly different.Comparsion with the Genbank sequence,the homology of nucleotide were 91.85%,93.78%,92.35% respectively,the homology of amino acid were93.15%,93.68%,92.47% respectively(2)? The concentration of G16 >250 ?g / m L in the MIC test inhibited the growth of Escherichia coli and Staphylococcus aureus;the concentration of GS16 > 15 ?g/mL significantly inhibited the growth of Escherichia coli,but had no inhibitory effect on Salmonella,Staphylococcus,Staphylococcus aureus.GC16 with a concentration of 62.5 ?g /mL had inhibitory effect on all four strains and significantly inhibited the growth of Escherichia coli and Staphylococcus aureus.G22 had no inhibitory effect on all four strains.G22 and GC16 significantly inhibited the growth of human esophageal cancer cells(EC109)and human renal cancer cells(X786-0),The growth inhibition rates against EC109 of G22 and GC16 with the concentration of 1 mg /mL were 88.21% and 57.21% respectively and its against X786-0 were 96.37% and 81.87% respectively.G16 and GS16 had no inhibitory activity to tumor cells.(3)?In animal experiments,pathological sections of PBS group found inflammatory cell infiltration,congestion,bleeding,acinar tissue incomplete,a large number of Cell mass in tissue gap,CFU counts of Staphylococcus aureus and TNF-? levels were significantly higher than the other three groups(P <0.05);peptide group and antibiotic group had no obvious pathological changes,breast of mice contained a small amount of milk,the epithelial cells and adipocytes of breast were eumorphism,occasionally isolated lymphocytes in the stroma,mammary tissue structure were basically complete,but the CFU count and TNF-? in the peptide group were significantly lower than those in the antibiotic group(P<0.05).Conclusion:Studies show that the GNLY gene is rich in polymorphisms in different breeds of sheep,and its secondary structure is predicted to contain 8 ?-helices and 6 LOOP structures;synthetic GNLY polypeptides have inhibitory activity against bacteria and tumor cells in vitro.The mouse model of mastitis has a good effect.This study laid the foundation for the research and development of sheep antibacterial peptide GNLY as an alternative antibacterial drug.