| The causes of bovine mastitis are complex and diverse,one of the important pathogens in which is Staphylococcus aureus(S.aureus).Currently,there are antibiotics,Chinese herbal medicines and inactivated vaccines to prevent or treatment bovine mastitis caused by S.aureus.However,excessive use of antibiotics will lead to resistance or even"resistance milk"which endanger public and human health.They are high dosage and high cost to use Chinese herbal medicines and not ideal to take advantage of inactivated vaccine.With the development of molecular biology and immunology,antibody biologics are attracting more and more researchers.In particular,small molecule antibody(scFv)not only retains the complete antigen binding site of the"Y"antibody,but also possesses the advantages of low molecular weight,low immunogenicity and strong tissue penetration ability.So,scFv(single-chain variable fragment)is a research focus for many scholars.In order to explore alternative antibiotics for the treatment of dairy cow mastitis,eight genes encoding scFv against S.aureus were inserted into pcDNA3.1(+)to construct the eukaryotic expression plasmid pcDNA3.1-scFv.Its prokaryotic expression product had been shown a obvious therapeutic effect in the previous experiment.Firstly,the recombinant eukaryotic plasmid was confirmed that began to express in mouse mammary gland tissue at first day and reached the expression peak at fifth day by Western blot.Then,the established mouse mastitis model of S.aureus infection was used to study the protective effect of scFv.Specific research content was that we injected the mixed eukaryotic expression plasmids through the fourth pair of mammary catheters into the mammary gland of mouse model of lactation mastitis.Therefore,scFv could inhibit the adhesion of bacteria to mammary epithelial cells of mice in order to protect mammary gland from S.aureus.Comparing with the negative control group,the number of bacteria in the mammary gland of scFvs treatment group was significantly reduced(p<0.05).The breast tissue was more integrated and the infiltration of inflammatory cells was lower.The levels of TNF-?,IL-6 and IL-17 were significantly lower than the negative control group(p<0.05).The result indicated that pcDNA3.1-scFvs might have a protective effect on mouse mastitis.In order to further verify the treatment of cow chronic mastitis by scFvs,seven dairy areas suffering from recessive mastitis were injected600?g mixed plasmids(pcDNA3.1-scFvs)twice at 48 h intervals by needleless syringe.After injection,the number of bacteria in the milk samples decreased significantly from 3~rdd day(p<0.05),and the CMT of 2breast areas were negative.So the cure rate was 28.57%.The CMT results of five milk samples were from++to+,which was judged as effective treatment.Consequently,the effective rate was 71.43%.The results indicated that pcDNA3.1-scFvs may have a certain degree of therapeutic effect on bovine subclinical mastitis.In this paper,milk,urine and manure samples from two cows No.11144 and No.14192 were also tested by PCR.No bands was found.It showed that the pcDNA3.1-scFvs in the mammary gland of the cows didn't enter the environment through milk,urine and manure.Therefore,pcDNA3.1-scFvs would not affect the environment.In conclusion,this study of pcDNA3.1-scFvs recombinant eukaryotic plasmid treatment of bovine mastitis,designed to not only provide a theoretical basis for gene therapy of bovine mastitis,but also explore a new way for the further study of alternative biological agents. |
PDF Full Text Request