Construction Antimicrobial Efficacy And Safety Evaluation Of Expression Plasmids Of Lyz And TAP Genes In Dairy Cattle

With the continuous improvement of dairy consumption,people pay more and more attention to the quality of milk.Dairy cow mastitis has become an important factor that seriously affects milk quality.Antibiotics are effective treatment methods,but its negative effects are increasingly prominent.Many problems,such as increased resistance of pathogenic bacteria and antibiotic residues in milk,lead to the decrease of milk yield and raw milk quality of dairy cows.Therefore,the development of a new alternative antibiotic product is of great significance to improve the economic benefits of pasture and milk quality.In this study,mammary gland specific expression plasmids pBLG-EGFP-N1-Lyz and pBLG-EGFP-N1-TAP were constructed by RT-PCR and homologous recombination.The above plasmids were transfected into primary mammary epithelial cells of dairy cows and mice at the peak lactation stage.Observation of green fluorescent protein pornography by fluorescence microscopy.Fluorescence quantitative PCR and Western Blot assay were used to verify the specific expression of the above plasmids at the level of mammary epithelial cells and in vivo mammary tissue.Analysis of antimicrobial effect of expressed products by cytotoxicity test.The safety of the recombinant plasmid was evaluated by weight-bearing test,hypoxia tolerance test,weight change weighing,blood biochemical index detection and organ index in mice.Specific expression of endogenous antimicrobial substances of Lyz and TAP in mammary gland tissue of dairy cows was achieved by recombinant plasmid.In order to prevent and cure mastitis and improve the quality of raw milk,the autoimmunity of dairy cows was improved.The main results are as follows:1.Construction of expression plasmid for treatment of dairy cow mastitisLyz and TAP gene coding sequences were amplified from dairy cow mammary epithelial cells by RT-PCR.Cloning it into a universal expression vector pEGFP-Nl carrying enhanced green fluorescent protein.Using homologous recombination technique,the original CMV promoter of pEGFP-N1 vector was replaced by the promoter of the mammary gland-specific expression of beta-lactoglobulin gene(BLG).Mammary gland specific expression plasmids pBLG-EGFP-N1-Lyz and pBLG-EGFP-N1-TAP were constructed.Agarose gel electrophoresis showed that the lysozyme gene(Lyz),tracheal antibacterial peptide gene(TAP)and beta lactobulin gene(BLG)promoter sequence were found in dairy cows.PCR amplification yielded bands consistent with expected fragment length.The bacterial fluid PCR detection showed that the target fragments had been linked to the expected position of the universal expression vector pEGFP-N1 of enhanced green fluorescent protein.Cloning and Sequencing of BLG Gene Promoter.BLAST alignment showed that the promoter sequence of the clone had 99%similarity with the 5'upstream sequence of the gene in the database.The promoter elements of GC-box,CAAT-box and TATA-box were found at 887 bp-892 bp,1188 bp-1191 BP and 2080-2085 BP respectively.It indicated that the selected fragment was the promoter fragment of BLG gene.The sequencing results of recombinant plasmids pBLG-EGFP-N1-Lyz and pBLG-EGFP-N1-TAP were consistent with the length of the target fragment.The target gene reading frame is correct and there is no wrong position.2.Expression Activity and Antibacterial Activity of Recombinant Plasmid CellsThe two recombinant plasmids were transfected into bovine primary mammary epithelial cells(bPMEC)and human renal epithelial cells(HEK293T)by liposome transfection.Fluorescence microscopy showed that only dairy cow primary mammary epithelial cells showed green fluorescence after transfection with two recombinant plasmids,but human kidney epithelial cells showed no color.Fluorescence quantitative PCR assay also showed that the expression of the two recombinant plasmids in primary mammary epithelial cells of dairy cows was significantly higher than that in non-transfected group,but not in human kidney epithelial cells.The survival rate of primary mammary epithelial cells transfected with recombinant plasmid was significantly higher than that of the control group without recombinant plasmid,and there was no significant difference compared with the control group without recombinant plasmid and Staphylococcus aureus.The cell death rate was significantly lower than that of the control group(P<0.01).3.Expression and safety evaluation of recombinant plasmid in mammary gland of miceThe gene was transfected in vitro by tail vein injection.The recombinant plasmids pBLG-EGFP-N1-Lyz and pBLG-EGFP-N1-TAP were transfected into mice at the peak lactation stage.The results of fluorescence quantitative PCR showed that the expression of TAP and Lyz genes in mouse mammary glands increased significantly.No expression of the target gene was detected in the heart,liver,spleen,lung and kidney of mice.Western Blot assay showed that protein products expressed by recombinant plasmid green fluorescent protein gene EGFP were detected in mammary tissue of mice transfected with recombinant plasmid,but not in non-transfected mammary tissue of mice.The results showed that the recombinant plasmid had no significant effect on the behavioral activity index,quality change,blood biochemical index and organ index of mice.In this study,two novel mammary gland specific expression plasmids were prepared.The expression and antimicrobial effect at cell level and in vivo level were also verified.It laid a foundation for the application of the plasmid in the prevention and treatment of cow mastitis in the future.It provides a new idea and method for prevention and treatment of dairy cow mastitis.To provide important theoretical basis and technical support for the development of new alternative antibiotic products.