Establishment Of Eukaryotic Expression System Of Dairy Cow Mastitis Etiology Gene Clf A,LPS And Hlb And Primary Evaluation Of Immune Protection Of Recombinant Clf A

Dairy cow mastitis was one of the most economic loss disease.After several decades' study,a comprehensive prevention and control system had been established in developed countries.But in most developing countries,it was still an economicconsuming disease due to backward disease-prevention-control theory and practice.To reduce the frequency,or eliminate of dairy cow mastitis were necessary to improve the herds' health standards,as well as animal welfare.Therefore,it was urgent to develop a universally effective vaccine.Amang the presently available options,the genetic engineering vaccine showed the most promising prospect.Because the etiology of dairy cow mastitis were varied,with multiple pathogen patterns from different geographical distribution.Vaccine products targeted at individual microbe received acceptable immune effect in certain herds,but could not guarantee enough protection to other hers located elsewhere.The gains could not balance the negative effect the vaccination process caused,or even cause stress and reduce the productivity of the herds.Simply combination of several bacteria could burden the immune system,triggering serious inflammation,or to the other extreme,inducing inadequate immune response due to insufficient antigen.This hopeless condition came to a halt when the genetic recombinant vaccine has come to spotlight,which allows multiple key antigens of different pathogens to be combined together without increasing the dosage of the vaccination,and at the same time,provide sufficient antigen to induce substantial protection.In this study,both conventional and molecular methods were adopted to isolate and determine the etiology of dairy cow mastitis of Gansu and around area.PCR technique was used to analyze the frequency of the genes that encoding antibiotic resistance and widely reported virulence genes contributing to their pathogenic process.Micro-dilution method was employed for phenotype analysis of antibiotic resistance of the isolates.Eukaryotic expression plasmids for the isolates' pathogenic genes were constructed and expressed in MCF-7 cells,and the gene products were VI purified and evaluated their potential as vaccine targets to reduce the losses caused by these isolates on Balb/c mice.Through carefully design and implement of the experiments,we safely made the following conclusions:After isolation and characterization of the 380 milk samples from Gansu and aground area,45 isolates were confirmed as Staphylococcus aureus,60 were confirmed Enterococcus spp.,13 were confirmed as Escherichia coli,nice were confirmed as Hafnia alvei and eight were Streptococcus spp.Micro-dilution method was adopted for the antibiotic resistance test of S.aureus to ten antibiotics.The results showed more than 80% S.aureus isolates were sensitive to ciprofloxacin,kanamycin and chloramphenicol,and were resistant to penicillin,ampicillin,erythromycin,and vancomycin.Besides,42% were resistant to tetracycline.Enterococcal isolates were resistant to penicillin,and 50% and 22% of the isolates were resistant to high level gentamycin and streptomycin.Most of the enterococcal isolates were sensitive to ampicillin,vancomycin and tetracycline.The E.coli isolates were remained sensitive to all the antimicrobial tested except 46% were resistant to tetracycline.The virulence profile of S.aureus were analyzed by PCR.The results showed the isolates were positive to Clf A,Fnb A,hlb,hld,hlg and seb,the proportion were 91%,88%,94%,91%,76% and 64% respectively.And the agr,luk S/E/M,hla,edin,eta,etb,tst and sea/c/d/e/g/i/j/n/o/m were not detected.There four eukaryotic expression plasmid were successfully constructed,the Clf A-pc DNA 3.1 V5-His B and hlb-pc DNA 3.1 V5-His B for S.aureus,the esppc DNA 3.1 V5-His B four Enterococcus and the LPS-pc DNA 3.1 V5-His B for E.coli.The Clf A-pc DNA 3.1 V5-His B was expressed in MCF-7 cell line and was confirmed by western blotting.The animal protection test showed that the recombinant Clf A protein immunized group triggered significantly higher interleukins,Ig Gs and IFN-? than the control group,indicating that the recombinant Clf A was qualified as a vaccine target for further development.The above results were based on practical facts,provided theoretical and practical basis for developing vaccine products for dairy cow mastitis.