| Cow mastitis is one of the most common and highest diseases in dairy industry,which can cause serious loss.Cow mastitis can not only reduce milk yield and milk quality,but also cause serious food safety problems.In the early stage,the prevention and treatment of mastitis was mainly the use of antibiotics peptide.However,due to the abuse and long-term use of antibiotics peptide,there are also other important problems in the treatment of dairy cow mastitis,such as bacterial resistance to antibiotics peptide,Effects on treatment and food safety caused by excessive antibiotic residues in milk.At present,in the treatment of dairy cow mastitis,the use of non-antibiotic substances is increasing,and non-antibiotic substances have gradually become the main drug for the treatment of dairy cow mastitis,and its treatment strategy has gradually replaced the previous antibiotic treatment.Antibiotics peptide(ABP)is a type of small molecule polypeptide that has a strong antibacterial effect and plays an important role in the body’s resistance to pathogen invasion.Antibiotics peptide have attracted widespread attention because of their unique antibacterial mechanism and wide spectrum and high efficiency antibacterial activity.At present,using genetic engineering method to express antimicrobial peptide gene to prevent and treat some diseases of poultry and livestock in animal husbandry is a hot spot in the application of antimicrobial peptide,and it is also one of the safe and effective methods to treat cow mastitis.In this study,the recombinant bovine LAP-CATHL2 antibiotic was cloned by overlapping extended PCR,and its prokaryotic,yeast and dairy cow mammary gland cell expression vectors were constructed.The recombinant cow LAP-CATHL2 antibiotic was expressed and purified,and the antibacterial spectrum and minimum antibacterial concentration of it were detected,and the sequence structure characteristics of it was analyzed.In addition,the effects of recombinant bovine LAP-CATHL2 antibiotics peptide on bovine mastitis were tested by constructing an animal model of bacterial cow mastitis.In this study,bovine beta-defensin LAP and bovine CATHL2 antibiotics peptide were cloned from bovine squamous epithelial cells and femoral bone marrow cells,respectively.And then,recombinant bovine LAP-CATHL2 antibiotics peptide were obtained by overlapping extended PCR cloning.In this study,recombinant cattle LAP-CATHL2 antibiotics peptide were cloned into p GEX-4T-1 vector,p PICZ αA vector,and p CMV-C-Flag vector contains an inserted regulatory sequence of αS1 casein as a promoter,respectively.And the prokaryotic expression vector,yeast expression vector and dairy cow mammary gland cell expression vector of recombinant bovine LAP-CATHL2 antibiotics peptide were constructed.These expression vectors were transferred to the Escherichia coli BL21,Pyretic yeast GS115 and purified primary cow mammary gland cells,respectively.The expression of recombinant bovine LAP-CATHL2 antibiotics peptide in these expression systems were induced and purified.The yield of recombinant bovine LAP-CATHL2 antibiotics peptide in the prokaryotic expression vector,yeast expression vector and dairy cow mammary gland cell expression vector is 2.83,1.16,and 0.114 mg/m L cultures,respectively.The antimicrobial spectra of recombinant bovine LAP-CATHL2 antibiotics peptide was analyzed by nine kinds of bacteria(Staphylococcus aureus ATCC25923,Escherichia coli ATCC25922,three kinds of Streptococcus(Sc.uberis,S.agalactiae,S.dysgalactiae equisimilis),Listeria monocytogenes NICPBP54002,Klebsiella spp,Bacillus cereus ATCC10987 and Nostoc)including gram-negative bacteria,gram-positive bacteria and fungi.The results showed that the recombinant bovine LAP-CATHL2 antibiotics peptide had a wide antimicrobial spectrum and had obvious antibacterial effect on gram-negative bacteria,gram-positive bacteria and fungi.Of the nine tested bacteria,the antibiotics peptide had the strongest antimicrobial resistance to Staphylococcus aureus,Escherichia coli,S.agalactiae and Nostoc.The Staphylococcus aureus,Escherichia coli,S.agalactiae and Nostoc were used to determine the minimum antibacterial concentration of recombinant bovine LAP-CATHL2 antibiotics peptide expressed by three different expression systems.The results showed that the antimicrobial activity of recombinant bovine LAP-CATHL2 antibiotics peptide expressed by prokaryotic expression system was the lowest,and the minimum antibacterial concentration for the four test bacteria was 500 μg/m L,250 μg/m L,250 μg/m L,and 500 μg/m L,respectively.The minimum antibacterial concentration of the recombinant bovine LAP-CATHL2 antibiotics peptide expressed by yeast expression system for four test bacteria was 125 μg/m L,62.5 μg/m L,62.5 μg/m L and 125 μg/m L,respectively.And the minimum antibacterial concentration of the recombinant bovine LAP-CATHL2 antibiotics peptide expressed by dairy cow mammary gland cell expression system for four test bacteria was 31.25 μg/m L,31.25 μg/m L,31.25 μg/m L and 62.5 μg/m L,respectively.The structure information of recombinant bovine LAP-CATHL2 antibiotics peptide such as hydrophilicity,transmembrane region,signal peptide structure,phosphorylation site,amino acid sequence primary structure,secondary structure and tertiary structure were analyzed using bioinformatics software and website prediction.The results show that the recombinant bovine LAP-CATHL2 antibiotics peptide have both hydrophilic and hydrophobic parts and belong to amphiphilic proteins.In addition,there are obvious transmembrane regions and multiple possible phosphorylation sites in the sequence.In the secondary and tertiary structures,the secondary structure of this antibiotic mainly consists of β-folding.It has multiple positive or reverse β-folding regions,and each β-folding region is connected by a loosely wound peptide chain at both ends.Using Staphylococcus aureus and Escherichia coli infection,an animal model of bacterial cow mastitis was constructed.The effects of recombinant bovine LAP-CATHL2 antibiotics peptide on bacterial cow mastitis were tested by breast perfusion and breast injection plasmid.The results showed that in the breast perfusion test,when the antibiotic concentration was 250 mg/m L and 10 m L antibiotic solution was perfusied daily in each breast area,the recombinant bovine LAP-CATHL2 antibiotics peptide expressed by yeast and dairy cow mammary gland cell expression system have a good therapeutic effect on bacterial cow mastitis,but the therapeutic effect of prokaryotic system expression was not obvious.When in the breast injection plasmid test,the injected plasmid from the dairy cow mammary gland cell expression system has a good preventive effect on bacterial cow mastitis but the injected plasmid from the prokaryotic and yeast expression system has no significant preventive effect.In summary,this study successfully cloned the recombinant bovine LAP-CATHL2 antibiotic,and constructed its prokaryotic,yeast and cow mammary gland cells to express plasmid.The recombinant bovine LAP-CATHL2 antibiotic was successfully induced and expressed in all three expression systems,and they all had broad spectrum antibacterial activity,but the antibacterial activity of recombinant antibiotics peptide expressed in prokaryotic expression system is the lowest.In addition,animal experiments have shown that recombinant antibiotics peptide expressed in vitro by yeast and dairy cow mammary cells have a significant effect on cow mastiti s caused by Staphylococcus aureus and Escherichia coli,but in the breast injection plasmid test,only the dairy cow mammary gland cell expression vector has a significant effect on the control of bacterial cow mastitis. |
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